Methods of treating lysosomal storage related diseases by gene therapy

ABSTRACT

Isolated nucleic acid-based vectors and lentivirus vectors, and methods of using those vectors to inhibit or prevent metabolic disorders in a mammal, are provided.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/057,410, filed Feb. 14, 2005, which is a continuation under 35 U.S.C.111(a) of PCT US/2003/025508, filed Aug. 13, 2003 and published on Jul.1, 2004 as WO 2004/055157 A2, which claims the benefit under 35 U.S.C.119(e) of U.S. Provisional Application Ser. No. 60/403,108, filed onAug. 13, 2002 and U.S. Provisional Application Ser. No. 60/403,586,filed Aug. 14, 2002, which are incorporated herein by reference.

STATEMENT OF GOVERNMENT RIGHTS

The invention was made at least in part with a grant from the Governmentof the United States of America (grant P01-HD32652 from the NationalInstitutes Health). The Government may have certain rights to theinvention.

BACKGROUND OF THE INVENTION

Many human genetic diseases are due to the deficiency of an enzyme orother protein. For example, the genetically determined deficiency of thelysosomal enzyme alpha-L-iduronidase results in the progressiveaccumulation of glycosaminoglycan substrates. In vitro evidence thatcells grown in culture can take up exogenously supplied enzymes from thesurrounding tissue culture medium led to the concept of in vivo enzymereplacement. For instance, enzyme replacement by intravenous infusionhas been demonstrated to be successful for adenosine deaminasedeficiency and for Gaucher disease, and some measures of efficacy havebeen found in human patients receiving weekly infusions of recombinanthuman alpha-L-iduronidase. However, these infusions must occur over aperiod of hours every week, and it is unclear if weekly administrationof the enzyme would be efficacious, even if treatments are started earlyin life. Moreover, it is likely that enzyme infusions will not preventprogressive mental retardation associated with particular proteindeficiencies.

Enzyme replacement may also be accomplished by transplantation ofgenetically normal cells and tissues, e.g., via bone marrowtransplantation for mucopolysaccharidosis (also known as Hurlersyndrome). For example, bone marrow transplantation was found to reducemany of the consequences of mucopolysaccharidosis type I and may preventprogression of mental retardation (Whitley et al., 1986). However,transplantation procedures which include the use of immunosuppressivemedications are associated with an increase in morbidity and mortality.

Enzyme replacement may also be accomplished via gene therapy, e.g., withviral vectors such as HIV-based vectors, ex vivo or in vivo. Lentiviralvectors are one type of viral vector which has been proposed as usefulfor mammalian gene therapy. HIV-based lentiviral vectors pseudotypedwith the envelope of another virus (most often the G protein of thevesicular stomatitis virus, VSVG) have become promising tools for genedelivery into nondividing cells. These vectors have been shown to becapable of transferring genes into a range of nonproliferative celltypes, including neurons, retinal cells, muscle cells, and hematopoieticpluripotent cells (Amado et al., 1999; Lever, 2000; Podsakoff, 2001)and, using local administration of those vectors, in vivo gene deliveryhas been accomplished in rat brain (Naldini et al., 1996; Blomer et al.,1997), liver and muscle (Kafri et al., 1997), retina (Miyoshi et al.,1999), and airway epithelia (Johnson et al., 2000).

However, the biosafety concerns surrounding HIV vectors have receivedconsiderable attention because of the pathogenic nature of HIV. Thus,efforts have been made to increase the safety of lentivirus vectors byminimizing the potential formation of replication-competent virus (RCR)via homologous recombination events. One strategy to reduce RCRformation has been to use nonoverlapping split-genome packagingconstructs that require multiple recombination events with the transfervector for RCR generation (Naldini et al., 1996; Reiser et al., 1996).Other strategies have focused on eliminating all unnecessary HIV readingframes from the system (Kim et al., 1998; Dull et al., 1998) ortruncating the 3N long terminal repeat (3N LTR) to generateself-inactivating HIV vectors (Miyoshi et al., 1998).

Lentiviral vectors have been a preferred vector for ex vivo modificationof hematopoietic (i.e., blood-forming) stem cells as lentiviral vectorsare likely capable of transducing such nondividing types of cells.Nevertheless, despite thousands of experiments attempting ex vivo genetransfer into hematopoietic stem cells, this vector-cell combination hasnot been successful in animal models of disease. Moreover, there hasnever been a successful clinical response in an animal using in vivolentiviral gene therapy, probably owing to insufficient delivery ofvector, or lack of expression of therapeutic protein, to thedisease-causing tissues or cells.

Thus, what is needed is an improved method to prevent, inhibit or treatmetabolic disorders characterized by a lack of, or a reduction in theamount of, an enzyme in a mammal.

SUMMARY OF THE INVENTION

The invention provides a lentivirus vector comprising a nucleic acidsegment encoding a gene product such as a protein, the absence orreduced levels of which are associated with a disorder in a mammal, adisorder including, but not limited to, a metabolic disorder, e.g., alysosomal storage disease, hemophilia, or adrenoleukodystrophy. Alentivirus includes ovine, caprine, equine, bovine and primate, e.g.,HIV-1, HIV-2 and SIV, lentiviruses. Also provided is a method in which arecombinant lentivirus comprising a nucleic acid segment encoding a geneproduct, the absence or reduced levels of which in a mammal areassociated with a disorder, is administered to a mammal having or atrisk of having such a disorder, in an amount effective to prevent,inhibit or treat at least one symptom associated with the disorder,e.g., a neurological symptom. In one embodiment, the recombinant virusis administered into a vascular compartment, e.g., intravenously, of themammal. Preferred amounts of virus include, but are not limited to,1×10³ to 1×10¹⁵ TU, e.g., 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴ or 1×10¹⁵ TU, although other amountsmaybe efficacious. Preferably, the mammal is a neonate or juvenile,although it is envisioned that adult mammals, and the developing embryoor fetus in utero, may also be treated.

In one embodiment, the recombinant lentivirus encodes a lysosomal enzymeand is administered in an amount which is effective to prevent, inhibitor treat a lysosomal storage disease in a mammal. Lysosomal storagediseases include, but are not limited to, mucopolysaccharidosisdiseases, for instance, mucopolysaccharidosis type I, e.g., Hurlersyndrome and the variants Scheie syndrome and Hurler-Scheie syndrome (adeficiency in alpha-L-iduronidase); Hunter syndrome (a deficiency ofiduronate-2-sulfatase); mucopolysaccharidosis type III, e.g., Sanfilipposyndrome (A, B, C or D; a deficiency of heparan sulfate sulfatase,N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminideN-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase);mucopolysaccharidosis type IV e.g., mucopolysaccharidosis type IV, e.g.,Morquio syndrome (a deficiency of galactosamine-6-sulfate sulfatase orbeta-galactosidase); mucopolysaccharidosis type VI, e.g., Maroteaux-Lamysyndrome (a deficiency of arylsulfatase B); mucopolysaccharidosis typeII; mucopolysaccharidosis type III (A, B, C or D; a deficiency ofheparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetylCoA:alpha-glucosaminide N-acetyl transferase orN-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IV(A or B; a deficiency of galactosamine-6-sulfatase andbeta-galatacosidase); mucopolysaccharidosis type VI (a deficiency ofarylsulfatase B); mucopolysaccharidosis type VII (a deficiency inbeta-glucuronidase); mucopolysaccharidosis type VIII (a, deficiency ofglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IX (adeficiency of hyaluronidase); Tay-Sachs disease (a deficiency in alphasubunit of beta-hexosaminidase); Sandhoff disease (a deficiency in bothalpha and beta subunit of beta-hexosaminidase); GM1 gangliosidosis (typeI or type II); Fabry disease (a deficiency in alpha galactosidase);metachromatic leukodystrophy (a deficiency of aryl sulfatase A); Pompedisease (a deficiency of acid maltase); fucosidosis (a deficiency offucosidase); alpha-mannosidosis (a deficiency of alpha-mannosidase);beta-mannosidosis (a deficiency of beta-mannosidase), ceroidlipofuscinosis, and Gaucher disease (types I, II and III; a deficiencyin glucocerebrosidase), as well as disorders such as Hermansky-Pudlaksyndrome; Amaurotic idiocy; Tangier disease; aspartylglucosaminuria;congenital disorder of glycosylation, type Ia; Chediak-Higashi syndrome;macular dystrophy, corneal, 1; cystinosis, nephropathic; Fanconi-Bickelsyndrome; Farber lipogranulomatosis; fibromatosis; geleophysicdysplasia; glycogen storage disease I; glycogen storage disease Ib;glycogen storage disease Ic; glycogen storage disease III; glycogenstorage disease IV; glycogen storage disease V; glycogen storage diseaseVI; glycogen storage disease VII; glycogen storage disease 0;immunoosseous dysplasia, Schimke type; lipidosis; lipase b;mucolipidosis II; mucolipidosis II, including the variant form;mucolipidosis IV; neuraminidase deficiency with beta-galactosidasedeficiency; mucolipidosis I; Niemann-Pick disease (a deficiency ofsphingomyelinase); Niemann-Pick disease without sphingomyelinasedeficiency (a deficiency of a npc1 gene encoding a cholesterolmetabolizing enzyme); Refsum disease; Sea-blue histiocyte disease;infantile sialic acid storage disorder; sialuria; multiple sulfatasedeficiency; triglyceride storage disease with impaired long-chain fattyacid oxidation; Winchester disease; Wolman disease (a deficiency ofcholesterol ester hydrolase); Deoxyribonuclease I-like 1 disorder;arylsulfatase E disorder; ATPase, H+ transporting, lysosomal, subunit 1disorder; glycogen storage disease IIb; Ras-associated protein rab9disorder; chondrodysplasia punctata 1, X-linked recessive disorder;glycogen storage disease VIII; lysosome-associated membrane protein 2disorder; Menkes syndrome; congenital disorder of glycosylation, typeIc; and sialuria. In particular, the invention is useful to prevent,inhibit or treat lysosomal storage diseases wherein the lysosomal enzymeis trafficked to the lysosome (within the cell and between cells) byspecific glycosylation. For most lysosomal enzymes and theircorresponding diseases, this would be by means of a terminalmannose-6-phosphate, however, it also includes terminal mannoseglycosylation, e.g., in the case of beta-glucocerebrosidase deficiencyresponsible for Gaucher disease. Thus, in one embodiment, the lentivirusvector of the invention is useful to prevent, inhibit or treat lysosomalstorage diseases including but are not limited to, mucopolysaccharidosisdiseases, for instance, mucopolysaccharidosis type I, e.g., Hurlersyndrome and the variants Scheie syndrome and Hurler-Scheie syndrome (adeficiency in alpha-L-iduronidase); mucopolysaccharidosis type II, e.g.,Hunter syndrome (a deficiency of iduronate-2-sulfatase);mucopolysaccharidosis type III, e.g., Sanfilippo syndrome (A, B, C or D;a deficiency of heparan sulfate sulfatase,N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminideN-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase);mucopolysaccharidosis type IV, e.g., Morquio syndrome (a deficiency ofgalactosamine-6-sulfate sulfatase or beta-galactosidase);mucopolysaccharidosis type VI, e.g., Maroteaux-Lamy syndrome (deficiencyof arylsulfatase B); mucopolysaccharidosis type VII, e.g., Sly syndrome(a deficiency in beta-glucuronidase); Tay-Sachs disease (a deficiency inalpha subunit of beta-hexosaminidase); Sandhoff disease (a deficiency inboth alpha and beta subunit of beta-hexosaminidase); GM1 gangliosidosis;Fabry disease (a deficiency in alpha-galactosidase); metachromaticleukodystrophy (a deficiency of aryl sulfatase A); Pompe disease (adeficiency of acid maltase); fucosidosis (a deficiency of fucosidase);alpha-mannosidosis (a deficiency of alpha-mannosidase);beta-mannosidosis (a deficiency of beta-mannosidase), ceroidlipofuscinosis, and Gaucher disease (types I, II and III; a deficiencyin glucocerebrosidase). As described herein, a single administration ofa lentivirus encoding alpha-L-iduronidase to newborn Hurler syndromemice resulted in normal patterns of behavior for the treated micerelative to untreated mice. Administration of the lentivirus to newbornslikely resulted in an increased efficiency of transduction which may inturn be due to the presence of cells that are more susceptible toinfection in the newborn. Early therapy, e.g., prenatal or in newborns,for metabolic disorders such as lysosomal storage diseases may thus beparticularly efficacious.

In one embodiment, a recombinant lentivirus encoding a lysosomal enzymeis administered to a mammal in an amount which is effective to increasethe level and/or activity of one or more lysosomal storage proteins,e.g., enzymes, and/or decrease skeletal deformity includingkyphoscoliosis, scoliosis, deformity or arthritis of the hip joints,contractures of the digits or larger joints at the knees, ankles, elbowsand shoulders, disfigurement of the face, recurrent and chronic earinfections, enlargement, dysfunction of an organ such as the liver,spleen or heart, obstruction of the coronary arteries causing myocardialinfarction, respiratory abnormality such as obstructive airway disease,reactive airway disease or pneumonia, brain or other nervous systemdamage, and/or dysfunction such as hydrocephalus, cranial nervecompression, hearing loss, blindness, spinal cord compression.

In one embodiment, a recombinant lentivirus encoding a lysosomal enzymeis administered to a mammal in an amount which is effective to increaselongevity, preserve intellect, e.g., measured by intelligence quotient(IQ), reduce ear infections, reduce skeletal deformity with improvedambulation, e.g., measured in a 6-minute walk test or other measurementsof endurance, reduce organ size (e.g., liver, spleen), improverespiratory function, e.g., measured by improved by spirometry,normalize of organ cellular architecture, e.g., observed by decreasedpathology (reduced lyosomal vacuolization or other microscopicpathology), decrease occlusion of the coronary arteries, reduce aberrantthickening of the meninges of the central nervous system, prevent orreduce hydrocephalus of the brain, decrease levels of pathologicsubstrates such as decreased glycosaminoglycan in the liver and othertissues, urine, or cerebrospinal fluid, and/or increase levels of adeficient enzyme such as alpha-L-iduronidase in liver tissue, whiteblood cells or plasma.

In one embodiment, a recombinant lentivirus encoding a lysosomal enzymeis administered intravenously to a mammal, e.g., a fetus (prenataldelivery), an infant (e.g., a human from birth to 2 years of age), achild (e.g., a human from over 2 years to 12 years or age), a juvenile(e.g., a human from over 12 years to 18 years of age), or an adult(e.g., a human older than 18 years of age).

In another embodiment, the invention provides a lentivirus vectorcomprising a nucleic acid sequence encoding a clotting factor, e.g.,Factor VIII or Factor IX, and a method to prevent, inhibit or treat amammal having or at risk of having the clotting disorder which employs arecombinant lentivirus comprising the vector. Preferably, therecombinant lentivirus is administered to a vascular compartment of themammal.

Further provided is a lentivirus vector comprising a nucleic acidsequence encoding an ABC protein such as a peroxisomal transportprotein, e.g., the X-ALD protein (ALDP), theadrenoleukodystrophy-related protein (ALDRP), the 70 kDa peroxisomalmembrane protein (PMP70), or a PMP70-related protein, and a method toprevent, inhibit or treat a mammal having or at risk of anadrenoleukodystrophy which employs a recombinant lentivirus comprisingthe vector. Preferably, the lentivirus is administered to a vascularcompartment of the mammal.

The invention also provides a recombinant lentivirus of the invention, ahost cell transfected with a lentivirus vector of the invention, e.g.,eukaryotic host cells including mammalian host cells such as human,non-human primate, canine, caprine, feline, bovine, equine, swine,ovine, rabbit or rodent cells, a host cell infected, e.g., ex vivo, witha recombinant lentivirus of the invention, and a method of expressing abiologically active protein in a cell which employs a lentivirus vectoror lentivirus of the invention which encodes the biologically activeprotein. A “biologically active” protein is a protein which hassubstantially the same activity, e.g., at least 80%, more preferably atleast 90%, the activity of a corresponding wild-type (functional)protein.

Also provided is a kit comprising a recombinant lentivirus of theinvention, e.g., a lyophilized or frozen preparation of recombinantlentivirus.

Mucopolysaccharidosis (MPS) type VII is an autosomal recessive lysosomalstorage disease resulting from deficiency of beta-glucuronidase due tomutations of the corresponding gene for beta-glucuronidase, GUSB. Asdescribed herein, a plasmid was constructed to express the human GUSBcDNA under the transcriptional regulation of a hybrid promoter-enhancer(CAGGS) containing the chicken beta-actin enhancer and CMV earlypromoter. Sleeping Beauty transposon IR sequences were included toexamine the potential for integration into the cell chromosome. Thistransposed plasmid pT-CAGGS-GUSB was administered by hydrodynamicinjection (i.e., intravenous infusion in a volume equal to 10% of bodyweight, over about 8-10 seconds) into the tail vein of mice ranging from4 to 23 weeks of age. The pT-CAGGS-GUSB plasmid was administered (25mcg/animal) alone (Group 1), or with transposase plasmid pSBI0, at atransposon:transposase molar ratio of 1:1 (Group 2), or 10:1 (Group 3).Forty-eight hours after injection, plasma beta-glucuronidase enzymaticactivity in treated MPS mice was markedly elevated (1,552-7,711nmol/ml/hr, n=14) in comparison to that of wild-type, untreated orsham-treated mice (9-15 nmol/ml/hr, n=6). In liver, beta-glucuronidaseactivity in treated MPS mice was also markedly elevated (1,860-6,185nmol/mg/hr, n=4) compared to normal levels (86-188 nmol/mg/hr). Notably,the liver tissue of MPS mice receiving pTCAGGS-GUSB stained uniformlypositive for beta-glucuronidase activity, including both Kupffer cellsand hepatocytes. One week after injection, plasma beta-glucuronidaseactivity was reduced relative to day 2 levels: Group 1, 59-93% of the2-day levels; Group 2, 21-36%; and Group 3, 33-63% (n=4 in each group).Beta-glucuronidase levels in the liver and spleen were 184-185nmol/mg/hr and 4,534-6,080, respectively, while levels in other organswere lower (heart 94-98, lung 49-65, kidney 59, and undetectable in thebrain). Two months after injection, beta-glucuronidase activity remainedat therapeutic levels in animals receiving pT-CAGGS-GUSB plasmid alone.Histochemical studies showed staining for beta-glucuronidase activitythroughout the liver and spleen. Remarkably, mice co-injected with pSBIOhad much lower levels of beta-glucuronidase activity. Morphometricanalysis of inclusion morphology demonstrated that clearing of hepaticlysosomal pathology was related to the level of beta-glucuronidase, andthat mice receiving pT-CAGGS-GUSB plasmid alone were completely clear ofpathology.

Thus, hydrodynamic infusion of the pT-CAGGS-GUSB transposon deliveredDNA to liver with marked increase in enzyme activity, with the highestlevels in blood ever achieved. GUSB enzymatic activity was foundthroughout the liver transiently reaching levels 10- to 1,000-fold ofnormal levels, levels which are above those that would be curative innewborns.

The results described herein with the lentivirus and plasmid vectors ofthe invention were surprising as the intravenous administration of othervectors did not show the extent of correction observed with thelentivirus and plasmid vectors. Moreover, PCR analysis of gonads, e.g.,testes, showed virtually no evidence of viral vector sequences,indicating a decreased risk for germ line transmission. Further, viralvector sequences were surprisingly detected in bone marrow stem cellsafter intravenous administration of a lentivirus vector of the inventionto a mammal and so those vectors are particularly useful for systemicexpression of therapeutic genes.

The invention provides a method to prevent, inhibit or treat a metabolicdisorder in a mammal via the hydrodynamic infusion of a plasmid encodinga gene product, the expression of which in the mammal prevents,inhibits, or treats one or more symptoms of the disorder. In oneembodiment, a fetus or neonate is infused via the umbilical cord with avector of the invention.

The invention provides a method to prevent, inhibit or treat a metabolicdisorder such as one characterized by the absence or reduced levels of alysosomal protein in a mammal. The method comprises administering to amammal, e.g., to a vascular compartment of a mammal having or at risk ofthe disorder an effective amount of an isolated nucleic acid moleculecomprising a nucleic acid sequence encoding the protein, e.g., abiologically active protein. In one embodiment, the nucleic acidmolecule comprises a promoter operably linked to the nucleic acidsequence.

The invention also provides isolated nucleic acid-based vectors toinhibit or treat metabolic disorders, e.g., lysosomal storage diseasesuch as mucopolysaccharidosis type I diseases, e.g., Hurler syndrome,mucopolysaccharidosis type II diseases, e.g., Hunter syndrome,mucopolysaccharidosis type II diseases, e.g., Sanfilippo syndrome,mucopolysaccharidoses type VII diseases, e.g., Sly disease, Fabrydisease, Gaucher disease as well as hemophilia, e.g., Factor VIII orfactor IX deficiency. Further provided is a method to prevent, inhibitor treat a metabolic disorder in a mammal which employs an isolatednucleic acid vector of the invention, e.g., in an amount effective toprevent, inhibit or treat at least one symptom associated with thedisorder, e.g., a neurological symptom associated with the disorder. Inone embodiment, the mammal is an adult. In one embodiment, the isolatednucleic acid vector of the invention is administered two or more timesto the mammal.

In another embodiment, the invention provides an isolated nucleic acidmolecule comprising a nucleic acid sequence encoding a clotting factor,e.g., Factor VIII or Factor IX, and a method to prevent, inhibit ortreat a mammal having or at risk of having the clotting disorder whichemploys a vector comprising the nucleic acid molecule. Preferably, thevector is administered to a vascular compartment of the mammal.

Further provided is an isolated nucleic acid molecule comprising anucleic acid sequence encoding an ABC protein such as a peroxisomaltransport protein, e.g., the X-ALD protein (ALDP), theadrenoleukodystrophy-related protein (ALDRP), the 70 kDa peroxisomalmembrane protein (PMP70), or a PMP70-related protein, and a method toprevent, inhibit or treat a mammal having or at risk of anadrenoleukodystrophy which employs a vector comprising the nucleic acidmolecule. Preferably, the lentivirus is administered to a vascularcompartment of the mammal.

The invention also provides an isolated nucleic acid molecule of theinvention, a host cell transfected with the isolated nucleic acidmolecule of the invention, e.g., eukaryotic host cells includingmammalian host cells such as human, non-human primate, canine, caprine,feline, bovine, equine, swine, ovine, rabbit or rodent cells, a hostcell transfected, e.g., ex vivo, with an isolated nucleic acid moleculeof the invention, and a method of expressing a biologically activeprotein in a cell which employs a vector comprising a nucleic acidmolecule of the invention which encodes the biologically active protein.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copes of this patent or patent application publication with colordrawings will be provided by the Office upon request and payment of thenecessary fee.

FIG. 1 shows the genetic map of a representative lentiviral vector forintravascular administration. LTR is the “long terminal repeat” of HIV-1virus; ψ⁺ is the packing signal; CMV refers to a promoter fromcytomegalovirus that regulates gene expression; PGK refers to aphosphoglycerate kinase promoter; the inverted triangle indicates amodification which results in a “self inactivating” vector; and IDUArefers to a cDNA sequence encoding the therapeutic proteinalpha-L-iduronidase.

FIG. 2 shows the increase in blood levels of therapeutic IDUA in MPS Imice treated by intravenous administration of an IDUA encodingrecombinant lentivirus contrasting the increase in those treated byintravenous administration of a control recombinant lentivirus. Therange of activity of IDUA in heterozygous IDUA transgenic mice is shownas a hatched band marked HET/HIGH HET/LOW.

FIG. 3 shows the therapeutic effect of intravenous administration of anIDUA encoding recombinant lentivirus on the facial pathology of a mousewith MPS I.

FIG. 4 shows preservation of normal behavior in MPS I mice due tointravenous administration of an IDUA encoding recombinant lentivirus.

FIG. 5 illustrates the pathology observed in untreated Hurler syndromemice (A), normal mice (B), and IDUA lentivirus-treated Hurler syndromemice (C). The sections were stained with a horseradish peroxidaseconjugated anti-alpha-GM2 ganglioside antibody.

FIG. 6 shows a schematic of pT-CAGGS-GUSB.

FIG. 7 shows histochemical visualization of beta-glucuronidase catalyticactivity in liver 48 hours after hydrodynamic infusion of pT-CAGGS-GUSBplasmid. Transfected cells stain intensively red. A) Untreated MPS VIImouse. B) Wild-type mouse showing normal levels of beta-glucuronidaseactivity. C) MPS VII mouse treated with pT-CAGGS-GUSB only. Dark redpunctuate spots are likely transfected cells expressing extremely highlevels of beta-glucuronidase enzyme while more diffusely red cells havetaken up enzyme by mannose-6-phosphate receptor-mediated endocytosis.(Representative views of 6 micron sections at 10× magnification.)

FIG. 8 shows data from two months after intravenous infusion ofpT-CAGSS-GUSB plasmid. Liver sections are stained for beta-glucuronidaseactivity (left) and with toluidine blue to visualize pathologiclysosomal vacuolization (right). A) and B) are MPS VII mouse untreated.C) and D) are wild-type mouse, untreated. E) and F) are MPS VII mousetreated with pT-CAGGS-GUSB alone. G) and H) are MPS VII mouse treated byco-injection of pT-CAGGS-GUSB and pSB plasmids in equal amounts, a 1:1ratio. I) and J) represent an MPS VII mouse treated by co-injection ofpT-CAGGS-GUSB and pSB plasmids in a ratio of 10:1.

FIG. 9 provides data from mice studied at 2 months after treatment.There is a dose-related correspondence of hepatic lysosomal pathology(area of vacuolization) to the level of hepatic beta-glucuronidaseenzyme activity. Mice receiving pT-CAGGS-GUSB demonstrated high levelsof enzyme activity and complete clearance of lysosomal pathology, whilethose co-injected with pT-CAGGS-GUSB and pSB showed intermediate levelsof response.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Inducible expression system” includes a construct or combination ofconstructs that includes a nucleotide sequence encoding atransactivator, an inducible promoter that can be transcriptionallyactivated by the transactivator, and a nucleotide sequence of interestoperably linked to the inducible promoter. For example, an exemplaryinducible expression system of the invention includes a nucleotidesequence encoding a tetracycline operon regulatable transactivator (tTA)and a nucleotide sequence of interest operably linked to an induciblepromoter composed of a minimal promoter operably linked to at least onetetO sequence.

“Transactivator,” “transactivating factor,” or “transcriptionalactivator” includes a polypeptide that facilitates transcription from apromoter. Where the promoter is an inducible promoter, thetransactivator activates transcription in response to a specifictranscriptional signal or set of transcriptional signals. For example,in the inducible expression system of the invention, tTA is atransactivator that facilitates transcription from the inducible tetOpromoter when tTA is not bound to tetracycline.

“Tetracycline repressor protein,” “tetracycline repressor polypeptide,”“tetR polypeptide,” and “tetR protein” are used interchangeably hereinto include a polypeptide that exhibits both 1) specific binding totetracycline and/or tetracycline derivatives; and 2) specific binding totetO sequences when the tetR polypeptide is not bound by tetracycline ora tetracycline analog(s). “TetR polypeptide” is meant to include anaturally occurring (i.e., native) tetR polypeptide sequence andfunctional derivatives thereof.

“Transcriptional activation domain” includes a polypeptide sequence thatfacilitates transcriptional activation from a promoter. “Transcriptionalactivation domain” includes transcriptional activation domains derivedfrom the naturally occurring amino acid sequence of a transcriptionfactor as well as functional derivatives thereof.

“Envelope protein” includes a polypeptide that 1) can be incorporatedinto an envelope of a retrovirus; and 2) can bind target cells andfacilitate infection of the target cell by the RNA virus that itenvelops. “Envelope protein” is meant to include naturally occurring(i.e., native) envelope proteins and functional derivatives thereofthat 1) can form pseudotyped retroviral virions according to theinvention, and 2) exhibit a desired functional characteristic(s) (e.g.,facilitate viral infection of a desired target cell, and/or exhibit adifferent or additional biological activity). In general, envelopeproteins of interest in the invention include any viral envelope proteinthat can, in combination with a retroviral genome, retroviral Pol,retroviral Gag, and other essential retroviral components, form aretroviral particle. Such envelope proteins include retroviral envelopeproteins derived from any suitable retrovirus (e.g., an amphotropic,xenotropic, ecotropic or polytropic retrovirus) as well asnon-retroviral envelope proteins that can form pseudotype retroviralvirions (e.g., VSV G). Envelope proteins of particular interest include,but are not limited to, envelope protein of vesicular stomatitis virus(VSV G), HTLV-1, gibbon ape leukemia virus (GALV), Sindai virus,influenza virus, herpes virus, rhabdovirus, and rabies virus.

“Functional derivative of a polypeptide” includes an amino acid sequencederived from a naturally occurring polypeptide that is altered relativeto the naturally occurring polypeptide by virtue of addition, deletion,substitution, or other modification of the amino acid sequence.“Functional derivatives” contemplated herein exhibit the characteristicsof the naturally occurring polypeptide essential to the operation of theinvention. For example, by “functional derivative of tetR” is meant apolypeptide derived from tetR that retains both 1) tetracycline ortetracycline analog binding and 2) the ability to inhibittranscriptional activation by tTA when bound to tetracycline or ananalog thereof.

“Promoter” includes a minimal DNA sequence sufficient to directtranscription of a DNA sequence to which it is operably linked. The term“promoter” is also meant to encompass those promoter elements sufficientfor promoter-dependent gene expression controllable for cell-typespecific expression, tissue-specific expression, or inducible byexternal signals or agents; such elements may be located in the 5N or 3Nregions of the naturally occurring gene.

“Inducible promoter” includes a promoter that is transcriptionallyactive when bound to a transcriptional activator, which in turn isactivated under a specific condition(s), e.g., in the presence of aparticular chemical signal or combination of chemical signals thataffect binding of the transcriptional activator to the induciblepromoter and/r affect function of the transcriptional activator itself.For example, the transcriptional activator of the present invention,tTA, induces transcription from its corresponding inducible promoterwhen tetracycline is absent, i.e., tetracycline is not bound to tTA.

“Construct” includes a recombinant nucleotide sequence, generally arecombinant DNA molecule, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences. In general,“construct” is used herein to refer to a recombinant DNA molecule.

“Operably linked” includes a DNA sequence and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequence(s).

“Isolated” when used in relation to a nucleic acid, as in “isolatedoligonucleotide” or “isolated polynucleotide” includes a nucleic acidsequence that is identified and separated from at least one contaminantwith which it is ordinarily associated in its source. Thus, an isolatednucleic acid is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acids(e.g., DNA and RNA) are found in the state they exist in nature. Forexample, a given DNA sequence (e.g., a gene) is found on the host cellchromosome in proximity to neighboring genes; RNA sequences (e.g., aspecific mRNA sequence encoding a specific protein), are found in thecell as a mixture with numerous other mRNAs that encode a multitude ofproteins. However, isolated nucleic acid includes, by way of example,such nucleic acid in cells ordinarily expressing that nucleic acid wherethe nucleic acid is in a chromosomal location different from that ofnatural cells, or is otherwise flanked by a different nucleic acidsequence than that found in nature. The isolated nucleic acid oroligonucleotide may be present in single-stranded or double-strandedform. When an isolated nucleic acid or oligonucleotide is to be utilizedto express a protein, the oligonucleotide contains at a minimum, thesense or coding strand (i.e., the oligonucleotide may single-stranded),but may contain both the sense and anti-sense strands (i.e., theoligonucleotide may be double-stranded). In one embodiment, the isolatednucleic acid is one which is free of viral proteins, i.e., it is not aviral particle, and/or does not encode one or more viral proteins.

“Operatively inserted” refers to a nucleotide sequence of interest ispositioned adjacent a nucleotide sequence that directs transcription andtranslation of the introduced nucleotide sequence of interest (i.e.,facilitates the production of e.g., a polypeptide encoded by a DNA ofinterest).

By “packaging cell line” is meant a line of packaging cells selected fortheir ability to package defective retroviral vectors at a titer ofgenerally greater than 10³ virions per milliliter of tissue culturemedium, having less than 10 helper virus virions per milliliter oftissue culture medium, and capable of being passaged in tissue culturewithout losing their ability to package defective retroviral vectors.

“Transformation” includes a permanent or transient genetic change,preferably a permanent genetic change, induced in a cell followingincorporation of new DNA (i.e., DNA exogenous to the cell). Where thecell is a mammalian cell, a permanent genetic change is generallyachieved by introduction of the DNA into the genome of the cell.

“Target cell” is a cell(s) that is to be transformed using the methodsand compositions of the invention. Transformation may be designed tonon-selectively or selectively transform the target cell(s). In general,target cell as used herein means a eukaryotic cell that can be infectedby a VSV G pseudotyped retroviral vector according to the invention.

“Transformed cell” is a cell into which (or into an ancestor of which)has been introduced, by means of recombinant DNA techniques, a DNAmolecule encoding a gene product (e.g., RNA and/or protein) of interest(i.e., nucleic acid encoding a therapeutic cellular product).

“Nucleotide sequence of interest”, “gene of interest” or “DNA ofinterest” includes any nucleotide or DNA sequence that encodes a proteinor other molecule that is desirable for expression in a host cell (e.g.,for production of the protein or other biological molecule (e.g., atherapeutic cellular product) in the target cell). The nucleotidesequence of interest is generally operatively linked to other sequenceswhich are needed for its expression, .e.g., a promoter. In general, anucleotide sequence of interest present in the genome of a recombinantretroviral particle of the invention encodes any gene product ofinterest, usually a therapeutic gene product where the recombinantretroviral particle is to be used to transform cells in vivo (e.g., in agene therapy application in humans).

A “therapeutic gene product” includes a polypeptide, RNA molecule orother gene product that, when expressed in a target cell, provides adesired therapeutic effect, e.g., repair of a genetic defect in thetarget cell genome (e.g., by complementation), expression of apolypeptide having a desired biological activity, and/or expression ofan RNA molecule for antisense therapy (e.g., regulation of expression ofa endogenous or heterologous gene in the target cell genome).

By “subject” or “patient” is meant any subject for which celltransformation or gene therapy is desired, including humans, cattle,dogs, cats, guinea pigs, rabbits, mice, insects, horses, chickens, andany other genus or species having cells that can be infected with aviral vector having an envelope containing VSV G or other envelopedescribed herein.

A “transgenic organism” includes a non-human organism (e.g., single-cellorganisms (e.g., yeast), mammal, non-mammal (e.g., nematode orDrosophila)) having a non-endogenous (i.e., heterologous) nucleic acidsequence present as an extrachromosomal element in a portion f its cellsor stably integrated into its germ line DNA.

A “transgenic animal” includes a non-human animal, usually a mammal,having a non-endogenous (i.e., heterologous) nucleic acid sequencepresent as an extrachromosomal element in a portion of its cells orstably integrated into its germ line DNA (i.e., in the genomic sequenceof most or all of its heterologous nucleic acid is introduced into thegerm line of such transgenic animals by genetic manipulation of, forexample, embryos or embryonic stem cells of the host animal.

A “viral vector” includes a recombinant viral particle that accomplishestransformation of a target cell with a nucleotide sequence of interest.

A “virion,” “viral particle,” or “retroviral particle” includes a singlevirus minimally composed of an RNA genome, a viral polymerase, e.g., aPol protein (for reverse transcription of the RNA genome followinginfection), a viral glycoprotein Gag protein (structural protein presentin the nucleocapsid), and an envelope protein. As used herein, the RNAgenome of a retroviral or lentiviral particle is usually a recombinantRNA genome, e.g., contains an RNA sequence exogenous to the nativeretroviral genome and/or is defective in an endogenous retrovirallentiviral sequence (e.g., is defective in pol, gag, and/or env, and, asused herein, is normally defective in all three genes).

“Pseudotyped viral particle,” or “pseudotyped retroviral particle”includes a viral particle having an envelope protein that is from avirus other than the virus from which the RNA genome is derived. Forinstance, the envelope protein can be from a retrovirus of a speciesdifferent from a retrovirus from which the RNA genome is derived or froma non-retroviral virus (e.g., vesicular stomatitis virus (VSV)).Preferably, the envelope protein of the pseudotyped retroviral particleis VSV G.

By “VSV G” or “VSV G envelope protein” is meant the envelope protein ofvesicular stomatitis virus (VSV) or a polypeptide derived therefrom orrecombinant fusion polypeptide having a VSV G polypeptide sequence fusedto a heterologous polypeptide sequence, where the VSV G-derivedpolypeptide of recombinant fusion polypeptide can be contained in aviral envelope of a pseudotyped retroviral particle and retainsinfectivity for a desired target cell (e.g., a range of desiredeukaryotic cells, or a specific target cell of interest).

By “VSV G pseudotyped virus,” “VSV G pseudotyped retrovirus,” “VSV Gpseudotyped viral particle,” or “VSV G pseudotyped retroviral particle,”is meant a retrovirus having the envelope protein VSV G, e.g., either incombination with or substantially substituted for the endogenousretroviral envelope. Preferably, VSV G is present in the VSV Gpseudotyped viral envelope such that VSV G represents about 50% of theenvelope protein(s) present in the envelope, more preferably about 75%,even more preferably about 90% to about 95%, still more preferablygreater than about 95%, most preferably about 100% or such that VSV G issubstantially the only envelope protein present in the pseudotyped viralparticle envelope.

Vectors for Recombinant Lentivirus Production

The lentiviral genome and the proviral DNA have the three genes found inretroviruses: gag, pol and env, which are flanked by two long terminalrepeat (LTR) sequences. The gag gene encodes the internal structural(matrix, capsid and nucleocapsid) proteins; the pol gene encodes theRNA-directed DNA polymerase (reverse transcriptase), a protease and anintegrase; and the env gene encodes viral envelope glycoproteins. The 5′and 3′ LTR's serve to promote transcription and polyadenylation of thevirion RNA's. The LTR contains all other cis-acting sequences necessaryfor viral replication. Lentiviruses have additional genes including vif,vpr, tat, rev, vpu, nef and vpx (in HIV-1, HIV-2 and/or SIV).

Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site). If thesequences necessary for encapsidation (or packaging of retroviral RNAinto infectious virions) are missing from the viral genome, the cisdefect prevents encapsidation of genomic RNA. However, the resultingmutant remains capable of directing the synthesis of all virionproteins.

The invention provides a method of producing a recombinant lentiviruscapable of infecting a cell, e.g., non-dividing cell, comprisingtransfecting a suitable host cell with two or more vectors carrying thepackaging functions, namely gag, pol and env, as well as rev and tat. Aswill be disclosed hereinbelow, vectors lacking a functional tat gene aredesirable for certain applications. Thus, for example, a first vectorcan provide a nucleic acid encoding a viral gag and a viral pol andanother vector can provide a nucleic acid encoding a viral env toproduce a packaging cell. Introducing a vector providing a heterologousgene, herein identified as a transfer vector, into that packaging cellyields a producer cell which releases infectious viral particlescarrying the heterologous gene of interest.

Generally the vectors are plasmid-based or virus-based, and areconfigured to carry the essential sequences for incorporating foreignnucleic acid, for selection and for transfer of the nucleic acid into ahost cell. The gag, pol and env genes of the vectors of interest alsoare known in the art. Thus, the relevant genes are cloned into theselected vector and then used to transform the target cell of interest.

According to the above-indicated configuration of vectors andheterologous genes, the second vector can provide a nucleic acidencoding a viral envelope (env) gene. The env gene can be derived fromany virus, including retroviruses, e.g., lentiviruses, and heterologousviruses such as VSV. The env preferably is an envelope protein whichallows transduction of cells of human and other species.

It may be desirable to target the recombinant virus by linkage of theenvelope protein with an antibody or a particular ligand for targetingto a receptor of a particular cell-type. By inserting a sequence(including a regulatory region) of interest into the viral vector, alongwith another gene which encodes the ligand for a receptor on a specifictarget cell, for example, the vector is now target-specific. Retroviralvectors can be made target-specific by inserting, for example, aglycolipid or a protein. Targeting often is accomplished by using anantigen-binding portion of an antibody or a recombinant antibody-typemolecule, such as a single chain antibody, to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific methods to achieve delivery of aretroviral vector to a specific target.

Examples of retroviral-derived env genes include, but are not limitedto: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murinesarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV orMMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiencyvirus (HIV), Rous sarcoma virus (RSV), and env genes of amphotropicviruses. Other env genes such as Vesicular stomatitis virus (VSV)protein G (VSV G), that of hepatitis viruses and of influenza also canbe used.

The vector providing the viral env nucleic acid sequence is associatedoperably with regulatory sequences, e.g., a promoter or enhancer. Theregulatory sequence can be any eukaryotic promoter or enhancer,including for example, the Moloney murine leukemia viruspromoter-enhancer element, the human cytomegalovirus (CMV) enhancer orthe vaccinia P7.5 promoter. In some cases, such as the Moloney murineleukemia virus promoter-enhancer element, the promoter-enhancer elementsare located within or adjacent to the LTR sequences.

Preferably, the regulatory sequence is one which is not endogenous,i.e., it is heterologous, to the lentivirus from which the vector isbeing constructed. Thus, if the vector is being made from SIV, the SIVregulatory sequence found in the SIV LTR would be replaced by aregulatory element which does not originate from SIV.

While VSV G protein is a desirable env gene because VSV G confers broadhost range on the recombinant virus, VSV G can be deleterious to thehost cell. Thus, when a gene such as that for VSV G is used, it ispreferred to employ an inducible promoter system so that VSV Gexpression can be regulated to minimize host toxicity when VSV G isexpression is not required. For example, the tetracycline-regulatablegene expression system of Gossen et al. (1992) can be employed toprovide for inducible expression of VSV G when tetracycline is withdrawnfrom the transfected cell. Thus, the tet/VP16 transactivator is presenton a first vector and the VSV G coding sequence is cloned downstreamfrom a promoter controlled by tet operator sequences on another vector.

The heterologous nucleic acid sequence of interest, the transgene, islinked operably to a regulatory nucleic acid sequence. As used herein,the term “heterologous” nucleic acid sequence refers to a sequence thatoriginates from a foreign species, or, if from the same species, it maybe substantially modified from the original form. Alternatively, anunchanged nucleic acid sequence that is not expressed normally in a cellis a heterologous nucleic acid sequence.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. Preferably, the heterologous sequence islinked to a promoter, resulting in a chimeric gene. The heterologousnucleic acid sequence is preferably under control of either the viralLTR promoter-enhancer signals or of an internal promoter, and retainedsignals within the retroviral LTR can still bring about efficientexpression of the transgene.

The heterologous gene of interest can be any nucleic acid of interestwhich can be transcribed. Generally the foreign gene encodes apolypeptide. Preferably the polypeptide has some therapeutic benefit.The polypeptide may supplement deficient or nonexistent expression of anendogenous protein in a host cell. The polypeptide can confer newproperties on the host cell, such as a chimeric signalling receptor, seeU.S. Pat. No. 5,359,046. The artisan can determine the appropriatenessof a heterologous gene practicing techniques taught herein and known inthe art. For example, the artisan would know whether a heterologous geneis of a suitable size for encapsidation and whether the heterologousgene product is expressed properly.

The method of the invention may also be useful for neuronal, glial,fibroblast or mesenchymal cell transplantation, or “grafting”, whichinvolves transplantation of cells infected with the recombinantlentivirus of the invention ex vivo, or infection in vivo into thecentral nervous system or into the ventricular cavities or subdurallyonto the surface of a host brain. Such methods for grafting will beknown to those skilled in the art and are described in Neural Graftingin the Mammalian CNS, Bjorklund & Stenevi, eds. (1985).

For diseases due to deficiency of a protein product, gene transfer couldintroduce a normal gene into the affected tissues for replacementtherapy, as well as to create animal models for the disease usingantisense mutations. For example, it may be desirable to insert a FactorVIII or IX encoding nucleic acid into a lentivirus for infection of amuscle, spleen or liver cell.

The promoter sequence may be homologous or heterologous to the desiredgene sequence. A wide range of promoters may be utilized, including aviral or a mammalian promoter. Cell or tissue specific promoters can beutilized to target expression of gene sequences in specific cellpopulations. Suitable mammalian and viral promoters for the instantinvention are available in the art.

Optionally during the cloning stage, the nucleic acid construct referredto as the transfer vector, having the packaging signal and theheterologous cloning site, also contains a selectable marker gene.Marker genes are utilized to assay for the presence of the vector, andthus, to confirm infection and integration. The presence of a markergene ensures the selection and growth of only those host cells whichexpress the inserts. Typical selection genes encode proteins that conferresistance to antibiotics and other toxic substances, e.g., histidinol,puromycin, hygromycin, neomycin, methotrexate etc. and cell surfacemarkers.

The recombinant virus of the invention is capable of transferring anucleic acid sequence into a mammalian cell. The term, “nucleic acidsequence”, refers to any nucleic acid molecule, preferably DNA, asdiscussed in detail herein. The nucleic acid molecule may be derivedfrom a variety of sources, including DNA, cDNA, synthetic DNA, RNA orcombinations thereof. Such nucleic acid sequences may comprise genomicDNA which may or may not include naturally occurring introns. Moreover,such genomic DNA may be obtained in association with promoter regions,poly A sequences or other associated sequences. Genomic DNA may beextracted and purified from suitable cells by means well known in theart. Alternatively, messenger RNA (mRNA) can be isolated from cells andused to produce cDNA by reverse transcription or other means.

Preferably, the recombinant lentivirus produced by the method of theinvention is a derivative of human immunodeficiency virus (HIV). The envwill be derived from a virus other than HIV.

Thus, three or more vectors, e.g., in one or more plasmids, whichprovide all of the functions required for packaging of recombinantvirions, such as, gag, pol, env, tat and rev, can be employed to preparerecombinant lentivirus. As noted herein, tat may be deleted. There is nolimitation on the number of vectors which are utilized so long as thevectors are used to transform and to produce the packaging cell line toyield recombinant lentivirus.

The vectors are introduced via transfection or infection into thepackaging cell line. The packaging cell line produces viral particlesthat contain the vector genome. Methods for transfection or infectionare well known by those of skill in the art. After cotransfection of thepackaging vectors and the transfer vector to the packaging cell line,the recombinant virus is recovered from the culture media and titered bystandard methods used by those of skill in the art.

Thus, the packaging constructs can be introduced into human cell linesby calcium phosphate transfection, lipofection or electroporation,generally together with a dominant selectable marker encoding, forexample, neomycin resistance, DHFR, Gln synthetase or ADA, followed byselection in the presence of the appropriate drug and isolation ofclones. The selectable marker gene can be linked physically to thepackaging genes in the construct.

Stable cell lines wherein the packaging functions are configured to beexpressed by a suitable packaging cell are known. For example, see U.S.Pat. No. 5,686,279; and Ory et al. (1996), which describe packagingcells.

Zufferey et al. (1997) teach a lentiviral packaging plasmid whereinsequences 3′ of pol including the HIV-1 env gene are deleted. Theconstruct contains tat and rev sequences and the 3′ LTR is replaced withpoly A sequences. The 5′ LTR and psi sequences are replaced by anotherpromoter, such as one which is inducible. For example, a CMV promoter orderivative thereof can be used.

The packaging vectors of interest may contain additional changes to thepackaging functions to enhance lentiviral protein expression and toenhance safety. For example, all of the HIV sequences upstream of gagcan be removed. Also, sequences downstream of env can be removed.Moreover, steps can be taken to modify the vector to enhance thesplicing and translation of the RNA.

To provide a vector with an even more remote possibility of generatingreplication competent lentivirus, lentivirus packaging plasmids whereintat sequences, a regulating protein which promotes viral expressionthrough a transcriptional mechanism, are deleted functionally. Thus, thetat gene can be deleted, in part or in whole, or various point mutationsor other mutations can be made to the tat sequence to render the genenon-functional. An artisan can practice known techniques to render thetat gene non-functional.

The techniques used to construct vectors, and to transfect and to infectcells, are practiced widely in the art. Practitioners are familiar withthe standard resource materials which describe specific conditions andprocedures.

A lentiviral packaging vector is made to contain a promoter and otheroptional or requisite regulatory sequences as determined by the artisan,gag, pol, rev, env or a combination thereof, and with specificfunctional or actual excision of tat, and optionally other lentiviralaccessory genes.

Lentiviral transfer vectors (Naldini et al., 1996) have been used toinfect human cells growth-arrested in vitro and to transduce neuronsafter direct injection into the brain of adult rats. The vector wasefficient at transferring marker genes in vivo into the neurons and longterm expression in the absence of detectable pathology was achieved.Another version of the lentiviral vector in which the HIV virulencegenes env, vif, vpr, vpu and nef were deleted without compromising theability of the vector to transduce non-dividing cells, represents asubstantial improvement in the biosafety of the vector (Zufferey et al.,1997).

In transduced cells, the integrated lentiviral vector generally has anLTR at each termini. The 5′ LTR may cause accumulation of “viral”transcripts that may be the substrate of recombination, in particular inHIV-infected cells. The 3′ LTR may promote downstream transcription withthe consequent risk of activating a cellular protooncogene. The U3sequences comprise the majority of the HIV LTR. The U3 region containsthe enhancer and promoter elements that modulate basal and inducedexpression of the HIV genome in infected cells and in response to cellactivation. Several of the promoter elements are essential for viralreplication. Some of the enhancer elements are highly conserved amongviral isolates and have been implicated as critical virulence factors inviral pathogenesis. The enhancer elements may act to influencereplication rates in the different cellular target of the virus (Marthaset al., 1993). Also, enhancers in either LTR can activate transcriptionof neighboring genes.

As viral transcription starts at the 3′ end of the U3 region of the 5′LTR, this U3 region (including the promoter and enhancer) is notincluded in the viral mRNA, and a copy thereof from the 3′ LTR acts astemplate for the generation of the U3 region of both LTR's in thesubsequently integrated provirus. If the U3 region of the 3′ LTR isaltered in a retroviral vector construct so as to eliminate the promoterand enhancer, the vector RNA still is produced from the intact 5′ LTR inproducer cells, but cannot be regenerated in target cells. Transductionof such a vector results in the transcriptional inactivation of bothLTR's in the progeny virus. Thus, the retrovirus is self-inactivating(SIN) and those vectors are known as Sin transfer vectors.

There are, however, limits to the extent of the deletion at the 3′ LTR.First, the 5′ end of the U3 region serves another essential function invector transfer, being required for integration (terminaldinucleotide+att sequence). Thus, the terminal dinucleotide and the attsequence may represent the 5′ boundary of the U3 sequences which can bedeleted. In addition, some loosely defined regions may influence theactivity of the downstream polyadenylation site in the R region.Excessive deletion of U3 sequence from the 3′ LTR may decreasepolyadenylation of vector transcripts with adverse consequences both onthe titer of the vector in producer cells and the transgene expressionin target cells. On the other hand, limited deletions may not abrogatethe transcriptional activity of the LTR in transduced cells.

U3 deletions in a HIV LTR can span from nucleotide-418 of the U3 LTR tothe indicated position: SIN-78, SIN-45, SIN-36 and SIN-18. Lentiviralvectors with almost complete deletion of the U3 sequences from the 3′LTR were developed without compromising either the titer of vector inproducer cells or transgene expression in target cells. The mostextensive deletion (−418 to −18) extends as far as to the TATA box,therefore abrogating any transcriptional activity of the LTR intransduced cells. Thus, the lower limit of the 3′ deletion may extend asfar as including the TATA box. The deletion may be of the remainder ofthe U3 region up to the R region. Surprisingly, the average expressionlevel of the transgene is higher in cells transduced by the SIN vectorsas compared to more intact vectors.

The 5′ LTR of a transfer vector construct can be modified bysubstituting part or all of the transcriptional regulatory elements ofthe U3 region with heterologous enhancer/promoters. The changes weremade to enhance the expression of transfer vector RNA in producer cells;to allow vector production in the absence of the HIV tat gene; and toremove the upstream wild-type copy of the HIV LTR that can recombinewith the 3′ deleted version to “rescue” the above described SIN vectors.

Thus, vectors containing the above-described alterations at the 5′ LTR,5′ vectors, can find use as transfer vectors because of the sequences toenhance expression and in combination with packaging cells that do notexpress tat. Such 5′ vectors can also carry modifications at the 3′ LTRas discussed hereinabove to yield improved transfer vectors which havenot only enhanced expression and can be used in packaging cells that donot express tat but can be self-inactivating as well.

The transcription from the HIV LTR is highly dependent on thetransactivator function of the tat protein. In the presence of tat,often expressed by the core packaging construct existing in producercells, vector transcription from the HIV LTR is stimulated strongly. Asthat full-length “viral” RNA has a full complement of packaging signals,the RNA is encapsidated efficiently into vector particles andtransferred to target cells. The amount of vector RNA available forpackaging in producer cells is a rate-limiting step in the production ofinfectious vector.

The entire enhancer or the entire enhancer and promoter regions of the5′ LTR can be substituted with the enhancer or the enhancer and promoterof the human cytomegalovirus (CMV) or murine Rous sarcoma virus (RSV).

The high level of expression of the 5′ LTR modified transfer vector RNAobtained in producer cells in the absence of a packaging constructindicates the producing vector is functional in the absence of afunctional tat gene. Functional deletion of the tat gene as indicatedfor the packaging plasmid disclosed hereinabove would confer a higherlevel of biosafety to the lentiviral vector system given the number ofpathogenetic activities associated with the tat protein.

Exemplary Packaging Cell Lines

Pseudotyped lentiviral or retroviral particles can be produced byintroducing a defective, recombinant lentiviral genome into a packagingcell (e.g., by infection with defective retroviral particle, or by othermeans for introducing DNA into a target cell, such as conventionaltransformation techniques). The defective retroviral genome minimallycontains the long terminal repeats, the exogenous nucleotide sequence ofinterest to be transferred, and a packaging sequence (N). In general,the packaging cell provides the missing retroviral components essentialfor retroviral replication, integration, and encapsidation, and alsoexpresses a nucleotide sequence encoding the desired envelope protein.However, the packaging cell does not have all of the componentsessential for the production of retroviral particles. The nucleotidesequence(s) encoding the missing viral component(s) in the packagingcell can be either stably integrated into the packaging cell genome,and/or can be provided by a co-infecting helper virus.

The nucleotide sequences encoding the retroviral components and thelentiviral or retroviral RNA genome can be derived from any desiredlenti- or retrovirus (e.g., murine, simian, avian, or humanretroviruses). In general, the retroviral components can be derived fromany retrovirus that can form pseudotyped retroviral particles with thedesired envelope protein, e.g., VSV G. Where VSV G is the desiredenvelope protein, the retroviral components can be derived from MuLV,MoMLV, avian leukosis virus (ALV), human immunodeficiency virus (HIV),or any other retrovirus that can form pseudotyped virus with VSV G asthe only envelope protein or with VSV G and a relatively small amount ofretroviral envelope protein.

The present invention thus provides recombinant retroviral particles,particularly pseudotyped retroviral particles. Exemplary packaging celllines are derived from 293, HeLa, Cf2Th, D17, MDCK, or BHK cells.Retroviral particles are preferentially produced by inducibly expressingan envelope protein of interest (e.g., a retroviral envelope or theenvelope protein of vesicular stomatitis virus). Inducible expression ofthe envelope protein may be accomplished by operably linking an envelopeprotein-encoding nucleotide sequence to an inducible promoter (e.g., apromoter composed of a minimal promoter linked to at least one copy oftetO, the binding site for the tetracycline repressor (tetR) of theEscherichia coli tetracycline resistance operon Tn10). Expression fromthe inducible promoter is regulated by a transactivating factor,composed of a first ligand-binding domain that negatively regulatestranscription from the inducible promoter (e.g., a prokaryotictetracycline repressor polypeptide (tetR)). Transcription of theenvelope-encoding nucleotide sequence under control of the induciblepromoter is activated by a transactivator when tetracycline is absent.

The packaging cell line may comprise a first polynucleotide having anHIV genome operably linked to a first inducible promoter wherein the HIVgenome is defective for cis-acting elements, for self-replication andfor expression of functional Env protein; a second polynucleotideencoding a functional heterologous Env protein operably linked to asecond inducible promoter; and a third polynucleotide encoding aregulatable transcriptional activator controlling transcription from thefirst and second inducible promoters.

In one embodiment, the first, second and third polynucleotides arecontained in vectors. These polynucleotides can be contained in one ormore vectors, preferably plasmid vectors. In an exemplary packaging cellline, the first polynucleotide is contained in a first plasmid vectorand the second polynucleotide is contained in a second plasmid vector.The third polynucleotide encoding a regulatable transcriptionalactivator is exemplified herein as containing a minimal CMVimmediate-early gene promoter linked to seven tandem copies of thetetR-binding site replacing the CMV promoter (BglII/BamHI fragment). Asdiscussed herein, other viral envelopes and other indicator markers willbe known to those of skill in the art for use in the present invention.

In one aspect of the invention, one or more polynucleotides encodingretroviral accessory proteins, are included as part of the first orsecond polynucleotide constructs, for example. Accessory proteinsinclude vpr, vif, nef, vpx, tat, eve, and vpu.

Preferably, the transcriptional activator or transactivator can beexpressed at high levels in a eukaryotic cell without significantlyadversely affecting general cellular transcription in the host celltransactivator expression that is sufficient to facilitatetransactivation of the inducible promoter, but that is not detrimentalto the cell (e.g., is not toxic to the cell). “High Levels” can be alevel of expression that allows detection of the transactivator byWestern blot The transactivator can preferably be expressed in a widevariety of cell types, including mammalian and non-mammalian cells suchas, but not limited to, human, monkey, mouse, hamster, cow, insect,fish, and frog cells.

The transactivator can be expressed either in vivo or in vitro, andexpression of the transactivator can be controlled through selection ofthe promoter to which the nucleotide sequence encoding thetransactivator is operably linked. For example, the promoter can be aconstitutive promoter or an inducible promoter. Examples of suchpromoters include the human cytomegalovirus promoter IE (Boshart et al.,1985), ubiquitously expressing promoters such as HSV-Tk (McKnight etal., 1984) and ∃-actin promoters (e.g., the human ∃-actin promoter asdescribed by Ng et al., 1985).

For example, where the transactivator is a tetR polypeptide, theinducible promoter is preferably a minimal promoter containing at leastone tetO sequence, preferably at least 2 or more tandemly repeated tetOsequences, even more preferably at least 5 or more tandemly repeatedtetO sequences, more preferably at least 7 tandemly repeated tetOsequences or more. The minimal promoter portion of the induciblepromoter can be derived from any desired promoter, and is selectedaccording to tet cell line in which the inducible expression system isto be used. Where the cell is a mammalian cell, a preferred minimalpromoter is derived from CMV, preferably from the CMV immediate earlygene 1A. In addition, other inducible promoters could be employed, suchas the ecdysone-inducible promoters (Invitrogen Inc., San Diego, Calif.)or the lacZ inducible promoters.

The promoter of the transactivator can be a cell type-specific ortissue-specific promoter than preferentially facilitates transcriptionof the transactivator in a desired cell of tissue type. Exemplary celltype-specific and/or tissue-specific promoters include promoters such asalbumin (liver specific; Pinkert et al., 1987), lymphoid-specificpromoters (Calame et al., 1988); in particular promoters of T-cellreceptors (Winoto et al., 1989) and immunoglobulins (Banerji et al.,1983; Queen and Baltimore, 1983), neuron-specific promoters (e.g., theneurofilament promoter (Byrne et al., 1989), pancreas-specific promoters(Edlunch et al., 1985) or mammary gland-specific promoters (milk wheypromoter, U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166). Promoters for expression of the transactivator can also bedevelopmentally regulated promoters as the murine homeobox promoters(Kessel et al., 1990) or the ∀-fetoprotein promoter (Campes et al.,1989). The promoter can be used in combination with control regionsallowing integration site independent expression of the transactivator(Grosveld et al., 1987). Preferably, the promoter is constitutive in therespective cell types. For instance, the promoter is a CMV promoter,preferably a CMV immediate early gene promoter.

Isolated Nucleic Acid-Based Vectors of the Invention

The isolated nucleic acid-based vectors of the invention, e.g., thosewhich are not delivered in a viral particle and/or do not encode one ormore viral proteins but may comprise viral transcriptional and/ortranslational regulatory elements, include a heterologous nucleic acidsequence of interest optionally operably linked to a regulatory nucleicacid sequence. The heterologous gene of interest in the isolated nucleicacid-based vector of the invention can be any nucleic acid of interestwhich can be transcribed. Generally the foreign gene encodes apolypeptide. Preferably the polypeptide has some therapeutic benefit.The polypeptide may supplement deficient or nonexistent expression of anendogenous protein in a host cell.

It may be desirable to modulate the expression of a gene regulatingmolecule in a cell by the introduction of a molecule by the method ofthe invention. The term “modulate” envisions the suppression ofexpression of a gene when it is over-expressed or augmentation ofexpression when it is under-expressed.

The method of the invention may also be useful for neuronal, glial,fibroblast or mesenchymal cell transplantation, or “grafting”, whichinvolves transplantation of transfected cells into the central nervoussystem or into the ventricular cavities or subdurally onto the surfaceof a host brain. Such methods for grafting will be known to thoseskilled in the art and are described in Neural Grafting in the MammalianCNS, Bjorklund & Stenevi, eds. (1985).

For diseases due to deficiency of a protein product, gene transfer of anisolated nucleic acid-based vector of the invention could introduce anormal gene into the affected tissues for replacement therapy, as wellas to create animal models for the disease using antisense mutations.

The promoter sequence of an isolated nucleic acid-based vector of theinvention may be homologous or heterologous to the desired genesequence. A wide range of promoters may be utilized, including a viralor a mammalian promoter. Cell or tissue specific promoters can beutilized to target expression of gene sequences in specific cellpopulations. Suitable mammalian and viral promoters for the instantinvention are available in the art.

Optionally during the cloning stage, the nucleic acid construct referredto as the transfer vector also contains a selectable marker gene. Markergenes are utilized to assay for the presence of the vector, and thus, toconfirm infection and integration. The presence of a marker gene ensuresthe selection and growth of only those host cells which express theinserts. Typical selection genes encode proteins that confer resistanceto antibiotics and other toxic substances, e.g., histidinol, puromycin,hygromycin, neomycin, methotrexate etc. and cell surface markers.

Exemplary Disorders and Genes

The invention includes the use of a vector, e.g., a lentiviral vector,comprising any open reading frame encoding a gene product useful toprevent, inhibit or treat a disorder in a mammal characterized by thelack of, or reduced levels of, that gene product. For example, thedisorder may be characterized by the lack of, or reduced levels of oneor more lysosomal enzymes (see, e.g., enzymes described in FIG. 5 inU.S. Pat. No. 5,798,366, the disclosure of which is specificallyincorporated by reference herein). Exemplary disorders include GM1gangliosidosis, which is caused by a deficiency in β-galactosidase;Tay-Sachs disease, a GM2 gangliosidosis which is caused by a deficiencyof β-hexosaminidase A (acidic isozyme); Sandhoff disease, which iscaused by a deficiency of β-hexosaminidase A & B (acidic and basicisozymes); Fabry disease, which is caused by a deficiency inα-galactosidase; Hurler syndrome, which is caused by a deficiency ofalpha-L-iduronidase, mucopolysaccharidosis type VII, which is caused bya deficiency in beta-glucuronidase, and Gaucher disease, which is adeficiency in β-glucocerebrosidase, as well as Hunter syndrome (adeficiency of iduronate-2-sulfatase); Sanfilippo syndrome (a deficiencyof heparan sulfate sulfatase, N-acetylglucosaminidase); Morquio syndrome(a deficiency of galactosamine-6-sulfate sulfatase orbeta-galactosidase); Maroteaux-Lamy syndrome (a deficiency ofarylsulfatase B); mucopolysaccharidosis type II; mucopolysaccharidosistype III (A, B, C or D; a deficiency of heparan sulfate sulfatase,N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminideN-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase);mucopolysaccharidosis type IV (A or B; a deficiency ofgalactosamine-6-sulfatase and beta-galatacosidase);mucopolysaccharidosis type VI (a deficiency of arylsulfatase B);mucopolysaccharidosis type VII (a deficiency in beta-glucuronidase);mucopolysaccharidosis type VIII (a deficiency of glucosamine-6-sulfatesulfatase); mucopolysaccharidosis type IX (a deficiency ofhyaluronidase); Tay-Sachs disease (a deficiency in alpha subunit ofbeta-hexosaminidase); Sandhoff disease (a deficiency in both alpha andbeta subunit of beta-hexosaminidase); GM1 gangliosidosis (type I or typeII); Fabry disease (a deficiency in alpha galactosidase); metachromaticleukodystrophy (a deficiency of aryl sulfatase A); Pompe disease (adeficiency of acid maltase); fucosidosis (a deficiency of fucosidase);alpha-mannosidosis (a deficiency of alpha-mannosidase);beta-mannosidosis (a deficiency of beta-mannosidase), ceroidlipofuscinosis, and Gaucher disease (types I, II and III; a deficiencyin glucocerebrosidase), as well as disorders such as Hermansky-Pudlaksyndrome; Amaurotic idiocy; Tangier disease; aspartylglucosaminuria;congenital disorder of glycosylation, type Ia; Chediak-Higashi syndrome;macular dystrophy, corneal, 1; cystinosis, nephropathic; Fanconi-Bickelsyndrome; Farber lipogranulomatosis; fibromatosis; geleophysicdysplasia; glycogen storage disease I; glycogen storage disease Ib;glycogen storage disease Ic; glycogen storage disease III; glycogenstorage disease IV; glycogen storage disease V; glycogen storage diseaseVI; glycogen storage disease VII; glycogen storage disease 0;immunoosseous dysplasia, Schimke type; lipidosis; lipase b;mucolipidosis II; mucolipidosis II, including the variant form;mucolipidosis IV; neuraminidase deficiency with beta-galactosidasedeficiency; mucolipidosis I; Niemann-Pick disease (a deficiency ofsphingomyelinase); Niemann-Pick disease without sphingomyelinasedeficiency (a deficiency of npc1, a cholesterol metabolizing enzyme);Refsum disease; Sea-blue histiocyte disease; infantile sialic acidstorage disorder; sialuria; multiple sulfatase deficiency; triglyceridestorage disease with impaired long-chain fatty acid oxidation;Winchester disease; Wolman disease (a deficiency of cholesterolhydrolase); Deoxyribonuclease I-like 1 disorder; arylsulfatase Edisorder; ATPase, H+ transporting, lysosomal, subunit 1 disorder;glycogen storage disease IIb; Ras-associated protein rab9 disorder;chondrodysplasia punctata 1, X-linked recessive disorder; glycogenstorage disease VIII; lysosome-associated membrane protein 2 disorder;Menkes syndrome; congenital disorder of glycosylation, type Ic; andsialuria. In particular, the invention is useful to prevent, inhibit ortreat lysosomal storage diseases wherein the lysosomal enzyme istrafficked to the lysosome (within the cell and between cells) byspecific glycosylation.

For instance, Tay-Sachs disease results from mutations in the HexA gene,which encodes the alpha subunit of β-hexosaminidase, leading to adeficiency in the A isoenzyme. The A isoenzyme is responsible for thedegradation of GM2 ganglioside. When this enzyme is deficient in humans,GM2 ganglioside accumulates progressively and leads to severeneurological degeneration. In the mouse model of Tay-Sachs disease(generated by the targeted disruption of the HexA gene) (Sandhoff etal., 1989), the mice store GM2 ganglioside in a progressive fashion, butthe levels never exceed the threshold required to elicitneurodegeneration. In the mouse (but not in a human) a sialidase issufficiently abundant that it can convert GM2 to GA2 (asialo ganglioside2), which can then be catabolized by the hexosaminidase β isoenzyme.

Gaucher disease is the name given to a group of lysosomal storagedisorders caused by mutations in the gene that codes for an enzymecalled glucocerebrosidase (“GC”). Gaucher disease is caused bydeficiency of GC. All of the mutations in the gene alter the structureand function of the enzyme which lead to an accumulation of theundegraded glycolipid substrate glucosylceramide, also calledglucocerebroside, in cells of the reticuloendothelial system. Eachparticular mutation of the human GC gene leads to a clinical diseasecollectively known as Gaucher disease. These disorders are usuallyclassified into three types; type 1 (non-neuronopathic), type 2 (acuteneuronopathic) and type 3 (subacute neuronopathic), the type dependingon the presence and severity of neurologic involvement.

GC is a monomeric, membrane-associated, hydrophobic glycoprotein with amolecular weight of 65,000 daltons. Human GC contains 497 amino acidsand is translated as a precursor protein with a 19 amino acidhydrophobic signal peptide which directs its co-translational insertioninto the lumen of the endoplasmic reticulum-golgi-lysosome complex asreported by Erickson et al. (1985). GC acts at the acidic pH of thelysosome to hydrolyze beta-glucosidic linkages in complex lipidsubiquitously found in all membranes to form the byproducts of glucoseand ceramide. The catalytic activity of GC is increased in vitro bydetergents, lipids, and in vivo by a naturally occurring activator knownas sphingolipid activator protein-2 (SAP-2 or saposin C). See, Ho et al.(1971); O′Brian et al. (1988). While more than twenty mutations in thehuman GC gene are known, only two are common. See, Tsuji et al. (1988).The two common mutations account for approximately 70% of the mutantalleles, as reported by Firon et al. (1990). Mutant GC genes code foraberrant proteins that are either catalytically altered or unstable andrapidly disappear from the cell.

Although GC is deficient in all of a subject's cells, for unknownreasons, the accumulation of the substrate glucosylceramide occursvirtually only in macrophages. To correct the enzyme deficiency inmacrophages, two approaches have been used. The first treatment is basedallogeneic bone marrow transplantation, which results in therepopulation of affected tissues with enzyme-competent macrophages. See,Rappeport et al. (1986). The second approach to treatment which hasresulted in clinical improvement in Gaucher disease patients ismacrophage-targeted enzyme replacement. This treatment takes advantageof naturally occurring mannose receptors on macrophages and theexposition of accessible mannose receptors in the oligosaccharides ofglucocerebrosidase to efficiently deliver the enzyme to macrophages.See, Barranger (1989); Takasaki et al. (1984); and Furbish et al.,(1981). However, allogeneic bone marrow transplantation has associatedwith it morbidity and mortality risks that are unacceptable for manypatients. Further, HLA matched bone marrow donors do not exist for themajority of patients. As for macrophage-targeted enzyme replacement, itis currently an expensive and life-long therapy.

Hurler syndrome is an autosomal recessive disease resulting fromdeficient alpha-iduronidase enzymatic activity and the consequentsystemic accumulation of glycosaminoglycan (GAG) substrates. The diseaseis characterized by hepatosplenomegaly, severe skeletal involvement,progressive mental retardation, and is typically lethal in childhood.

To be an effective permanent treatment for any disease capable of beingtreated by gene therapy, the transfer and sustained expression of genesin cells important to the pathogenesis of the particular disease isrequired. Sufficient and long term expression of a transduced gene inthe progeny of transduced cells, e.g., transduced stem cells such aspluripotent bone marrow stem cells, for example, using a lentivirus,could correct the deficiency of the enzyme in many if not all relevantcell types.

Dosages, Formulations and Routes of Administration of the Agents of theInvention

The therapeutic agents of the invention are preferably administered soas to achieve beneficial results. The amount administered will varydepending on various factors including, but not limited to, the agentchosen, the disease, whether prevention or treatment is to be achieved,and if the agent is modified for bioavailability and in vivo stability.

Administration of sense or antisense nucleic acid molecule may beaccomplished through the introduction of cells transformed with anexpression cassette comprising the nucleic acid molecule (see, forexample, WO 93/02556) or the administration of the nucleic acid molecule(see, for example, Felgner et al., U.S. Pat. No. 5,580,859, Pardoll etal., Immunity, 3, 165 (1995); Stevenson et al., Immunol. Rev., 145, 211(1995); Moiling, J. Mol. Med., 75, 242 (1997); Donnelly et al., Ann.N.Y. Acad. Sci., 772, 40 (1995); Yang et al., Mol. Med. Today. 2, 476(1996); Abdallah et al., Biol. Cell, 85, 1 (1995)). Pharmaceuticalformulations, dosages and routes of administration for nucleic acids aregenerally disclosed, for example, in Felgner et al., supra. Nucleic acidmolecules may be complexed with polyethyleneimine, polylysine orcationic lipids such as DOTMA, DOTAP, DOGS, or DC-Chol(N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium chloride, DOTAP;N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium chloride, DOTMA;3β-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol, DC-Chol) In oneembodiment, DNA is delivered under pressure into the hepaticcirculation.

The amount of therapeutic agent administered is selected to treat aparticular indication. The therapeutic agents of the invention are alsoamenable to chronic use for prophylactic purposes, preferably bysystemic administration.

Administration of the therapeutic agents in accordance with the presentinvention may be continuous or intermittent, depending, for example,upon the recipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of the agents of theinvention may be essentially continuous over a preselected period oftime or may be in a series of spaced doses. Both local and systemicadministration is contemplated.

One or more suitable unit dosage forms comprising the therapeutic agentsof the invention, which, as discussed below, may optionally beformulated for sustained release, can be administered by a variety ofroutes including oral, or parenteral, including by rectal, buccal,vaginal and sublingual, transdermal, subcutaneous, intravenous,intramuscular, intraperitoneal, intrathoracic, intrapulmonary andintranasal routes. The formulations may, where appropriate, beconveniently presented in discrete unit dosage forms and may be preparedby any of the methods well known to pharmacy. Such methods may includethe step of bringing into association the therapeutic agent with liquidcarriers, solid matrices, semi-solid carriers, finely divided solidcarriers or combinations thereof, and then, if necessary, introducing orshaping the product into the desired delivery system.

When the therapeutic agents of the invention are prepared for oraladministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. The total active ingredients in suchformulations comprise from 0.1 to 99.9% by weight of the formulation. By“pharmaceutically acceptable” it is meant the carrier, diluent,excipient, and/or salt must be compatible with the other ingredients ofthe formulation, and not deleterious to the recipient thereof. Theactive ingredient for oral administration may be present as a powder oras granules; as a solution, a suspension or an emulsion; or inachievable base such as a synthetic resin for ingestion of the activeingredients from a chewing gum. The active ingredient may also bepresented as a bolus, electuary or paste.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, douches, lubricants, foams orsprays containing, in addition to the active ingredient, such carriersas are known in the art to be appropriate. Formulations suitable forrectal administration may be presented as suppositories.

Pharmaceutical formulations containing the therapeutic agents of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. For example, the agent can beformulated with common excipients, diluents, or carriers, and formedinto tablets, capsules, suspensions, powders, and the like. Examples ofexcipients, diluents, and carriers that are suitable for suchformulations include the following fillers and extenders such as starch,sugars, mannitol, and silicic derivatives; binding agents such ascarboxymethyl cellulose, HPMC and other cellulose derivatives,alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing agents suchas glycerol; disintegrating agents such as calcium carbonate and sodiumbicarbonate; agents for retarding dissolution such as paraffin;resorption accelerators such as quaternary ammonium compounds; surfaceactive agents such as cetyl alcohol, glycerol monostearate; adsorptivecarriers such as kaolin and bentonite; and lubricants such as talc,calcium and magnesium stearate, and solid polyethyl glycols.

For example, tablets or caplets containing the agents of the inventioncan include buffering agents such as calcium carbonate, magnesium oxideand magnesium carbonate. Caplets and tablets can also include inactiveingredients such as cellulose, pregelatinized starch, silicon dioxide,hydroxy propyl methyl cellulose, magnesium stearate, microcrystallinecellulose, starch, talc, titanium dioxide, benzoic acid, citric acid,corn starch, mineral oil, polypropylene glycol, sodium phosphate, andzinc stearate, and the like. Hard or soft gelatin capsules containing anagent of the invention can contain inactive ingredients such as gelatin,microcrystal line cellulose, sodium lauryl sulfate, starch, talc, andtitanium dioxide, and the like, as well as liquid vehicles such aspolyethylene glycols (PEGs) and vegetable oil. Moreover, enteric coatedcaplets or tablets of an agent of the invention are designed to resistdisintegration in the stomach and dissolve in the more neutral toalkaline environment of the duodenum.

The therapeutic agents of the invention can also be formulated aselixirs or solutions for convenient oral administration or as solutionsappropriate for parenteral administration, for instance byintramuscular, subcutaneous or intravenous routes.

The pharmaceutical formulations of the therapeutic agents of theinvention can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The active ingredients may takesuch forms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable vehicles andadjuvants which are well known in the prior art. It is possible, forexample, to prepare solutions using one or more organic solvent(s) thatis/are acceptable from the physiological standpoint, chosen, in additionto water, from solvents such as acetone, ethanol, isopropyl alcohol,glycol ethers such as the products sold under the name “Dowanol”,polyglycols and polyethylene glycols, C₁-C₄ alkyl esters of short-chainacids, preferably ethyl or isopropyl lactate, fatty acid triglyceridessuch as the products marketed under the name “Miglyol”, isopropylmyristate, animal, mineral and vegetable oils and polysiloxanes.

The compositions according to the invention can also contain thickeningagents such as cellulose and/or cellulose derivatives. They can alsocontain gums such as xanthan, guar or carbo gum or gum arabic, oralternatively polyethylene glycols, bentones and montmorillonites, andthe like.

It is possible to add, if necessary, an adjuvant chosen fromantioxidants, surfactants, other preservatives, film-forming,keratolytic or comedolytic agents, perfumes and colorings. Also, otheractive ingredients may be added, whether for the conditions described orsome other condition.

For example, among antioxidants, t-butylhydroquinone, butylatedhydroxyanisole, butylated hydroxytoluene and α-tocopherol and itsderivatives may be mentioned. The galenical forms chiefly conditionedfor topical application take the form of creams, milks, gels, dispersionor microemulsions, lotions thickened to a greater or lesser extent,impregnated pads, ointments or sticks, or alternatively the form ofaerosol formulations in spray or foam form or alternatively in the formof a cake of soap.

Additionally, the agents are well suited to formulation as sustainedrelease dosage forms and the like. The formulations can be soconstituted that they release the active ingredient only or preferablyin a particular part of the intestinal or respiratory tract, possiblyover a period of time. The coatings, envelopes, and protective matricesmay be made, for example, from polymeric substances, such aspolylactide-glycolates, liposomes, microemulsions, microparticles,nanoparticles, or waxes. These coatings, envelopes, and protectivematrices are useful to coat indwelling devices, e.g., stents, catheters,peritoneal dialysis tubing, and the like.

The therapeutic agents of the invention can be delivered via patches fortransdermal administration. See U.S. Pat. No. 5,560,922 for examples ofpatches suitable for transdermal delivery of a therapeutic agent.Patches for transdermal delivery can comprise a backing layer and apolymer matrix which has dispersed or dissolved therein a therapeuticagent, along with one or more skin permeation enhancers. The backinglayer can be made of any suitable material which is impermeable to thetherapeutic agent. The backing layer serves as a protective cover forthe matrix layer and provides also a support function. The backing canbe formed so that it is essentially the same size layer as the polymermatrix or it can be of larger dimension so that it can extend beyond theside of the polymer matrix or overlay the side or sides of the polymermatrix and then can extend outwardly in a manner that the surface of theextension of the backing layer can be the base for an adhesive means.Alternatively, the polymer matrix can contain, or be formulated of, anadhesive polymer, such as polyacrylate or acrylate/vinyl acetatecopolymer. For long-term applications it might be desirable to usemicroporous and/or breathable backing laminates, so hydration ormaceration of the skin can be minimized.

Examples of materials suitable for making the backing layer are films ofhigh and low density polyethylene, polypropylene, polyurethane,polyvinylchloride, polyesters such as poly(ethylene phthalate), metalfoils, metal foil laminates of such suitable polymer films, and thelike. Preferably, the materials used for the backing layer are laminatesof such polymer films with a metal foil such as aluminum foil. In suchlaminates, a polymer film of the laminate will usually be in contactwith the adhesive polymer matrix.

The backing layer can be any appropriate thickness which will providethe desired protective and support functions. A suitable thickness willbe from about 10 to about 200 microns.

Generally, those polymers used to form the biologically acceptableadhesive polymer layer are those capable of forming shaped bodies, thinwalls or coatings through which therapeutic agents can pass at acontrolled rate. Suitable polymers are biologically and pharmaceuticallycompatible, nonallergenic and insoluble in and compatible with bodyfluids or tissues with which the device is contacted. The use of solublepolymers is to be avoided since dissolution or erosion of the matrix byskin moisture would affect the release rate of the therapeutic agents aswell as the capability of the dosage unit to remain in place forconvenience of removal.

Exemplary materials for fabricating the adhesive polymer layer includepolyethylene, polypropylene, polyurethane, ethylene/propylenecopolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetatecopolymers, silicone elastomers, especially the medical-gradepolydimethylsiloxanes, neoprene rubber, polyisobutylene, polyacrylates,chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinylacetate copolymer, crosslinked polymethacrylate polymers (hydrogel),polyvinylidene chloride, poly(ethylene terephthalate), butyl rubber,epichlorohydrin rubbers, ethylenevinyl alcohol copolymers,ethylene-vinyloxyethanol copolymers; silicone copolymers, for example,polysiloxane-polycarbonate copolymers, polysiloxane, polyethylene oxidecopolymers, polysiloxane-polymethacrylate copolymers,polysiloxane-alkylene copolymers (e.g., polysiloxane-ethylenecopolymers), polysiloxane-alkylenesilane copolymers (e.g.,polysiloxane-ethylenesilane-copolymers), and the like; cellulosepolymers, for example methyl or ethyl cellulose, hydroxy propyl methylcellulose, and cellulose esters; polycarbonates;polytetrafluoroethylene; and the like.

Preferably, a biologically acceptable adhesive polymer matrix should beselected from polymers with glass transition temperatures below roomtemperature. The polymer may, but need not necessarily, have a degree ofcrystallinity at room

temperature. Cross-linking monomeric units or sites can be incorporatedinto such polymers. For example, cross-linking monomers can beincorporated into polyacrylate polymers, which provide sites forcross-linking the matrix after dispersing the therapeutic agent into thepolymer. Known cross-linking monomers for polyacrylate polymers includepolymethacrylic esters of polyols such as butylene diacrylate anddimethacrylate, trimethylol propane trimethacrylate and the like. Othermonomers which provide such sites include allyl acrylate, allylmethacrylate, diallyl maleate and the like.

Preferably, a plasticizer and/or humectant is dispersed within theadhesive polymer matrix. Water-soluble polyols are generally suitablefor this purpose. Incorporation of a humectant in the formulation allowsthe dosage unit to absorb moisture on the surface of skin which in turnhelps to reduce skin irritation and to prevent the adhesive polymerlayer of the delivery system from failing.

Therapeutic agents released from a transdermal delivery system must becapable of penetrating each layer of skin. In order to increase the rateof permeation of a therapeutic agent, a transdermal drug delivery systemmust be able in particular to increase the permeability of the outermostlayer of skin, the stratum corneum, which provides the most resistanceto the penetration of molecules. The fabrication of patches fortransdermal delivery of therapeutic agents is well known to the art.

For administration to the upper (nasal) or lower respiratory tract byinhalation, the therapeutic agents of the invention are convenientlydelivered from an insufflator, nebulizer or a pressurized pack or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof the therapeutic agent and a suitable powder base such as lactose orstarch. The powder composition may be presented in unit dosage form in,for example, capsules or cartridges, or, e.g., gelatine or blister packsfrom which the powder may be administered with the aid of an inhalator,insufflator or a metered-dose inhaler.

For intra-nasal administration, the therapeutic agent may beadministered via nose drops, a liquid spray, such as via a plasticbottle atomizer or metered-dose inhaler. Typical of atomizers are theMistometer (Wintrop) and the Medihaler (Riker).

The local delivery of the therapeutic agents of the invention can alsobe by a variety of techniques which administer the agent at or near thesite of disease. Examples of site-specific or targeted local deliverytechniques are not intended to be limiting but to be illustrative of thetechniques available. Examples include local delivery catheters, such asan infusion or indwelling catheter, e.g., a needle infusion catheter,shunts and stents or other implantable devices, site specific carriers,direct injection, or direct applications.

For topical administration, the therapeutic agents may be formulated asis known in the art for direct application to a target area.Conventional forms for this purpose include wound dressings, coatedbandages or other polymer coverings,

ointments, creams, lotions, pastes, jellies, sprays, and aerosols, aswell as in toothpaste and mouthwash, or by other suitable forms, e.g.,via a coated condom. Ointments and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Lotions may be formulated with anaqueous or oily base and will in general also contain one or moreemulsifying agents, stabilizing agents, dispersing agents, suspendingagents, thickening agents, or coloring agents. The active ingredientscan also be delivered via iontophoresis, e.g., as disclosed in U.S. Pat.Nos. 4,140,122; 4,383,529; or 4,051,842. The percent by weight of atherapeutic agent of the invention present in a topical formulation willdepend on various factors, but generally will be from 0.01% to 95% ofthe total weight of the formulation, and typically 0.1-25% by weight.

When desired, the above-described formulations can be adapted to givesustained release of the active ingredient employed, e.g., bycombination with certain hydrophilic polymer matrices, e.g., comprisingnatural gels, synthetic polymer gels or mixtures thereof.

Drops, such as eye drops or nose drops, may be formulated with anaqueous or non-aqueous base also comprising one or more dispersingagents, solubilizing agents or suspending agents. Liquid sprays areconveniently delivered from pressurized packs. Drops can be deliveredvia a simple eye dropper-capped bottle, or via a plastic bottle adaptedto deliver liquid contents dropwise, via a specially shaped closure.

The therapeutic agent may further be formulated for topicaladministration in the mouth or throat. For example, the activeingredients may be formulated as a lozenge further comprising a flavoredbase, usually sucrose and acacia or tragacanth; pastilles comprising thecomposition in an inert base such as gelatin and glycerin or sucrose andacacia; mouthwashes comprising the composition of the present inventionin a suitable liquid carrier; and pastes and gels, e.g., toothpastes orgels, comprising the composition of the invention.

The formulations and compositions described herein may also containother ingredients such as antimicrobial agents, or preservatives.Furthermore, the active ingredients may also be used in combination withother therapeutic agents.

The invention is further described by the following non-limitingexamples.

Example I Materials and Methods

Production and concentration of lentiviral vector. The lentiviral vectorwas generated from a first-generation tetracycline-inducibleVSVG-pseudotyped lentiviral packaging cell line SODk1-CGFI (Kafri etal., 1999). The vector (HRNcmvGFP) contained a humanized red-shift GFPgene driven by a human cytomegalovirus immediate-early promoter. Thevector producer cells were maintained in Dulbecco's modified Eagle'smedium (DMEM) (Life Technologies, Inc., Gaithersburg, Md.) containing10% tetracycline-free FCS (Clontech, Palo Alto, Calif.) and 0.7 :g/mldoxycycline (Sigma, St. Louis, Mo.). Induction of cells was initiated bysplitting cells into polylysine (0.01% solution; Sigma) precoated platesin the absence of doxycycline. The cells were washed twice with PBS(Life Technologies Inc.) and fed daily with doxycycline-free mediumcontaining 5 mM sodium butyrate (Sigma). Vector-containing medium wascollected 3 and 4 days after induction and filtered through a 0.2 :mpore filter. The vector stocks were further concentrated byultracentrifugation at 50,000 g for 2 hours (Beckman SW-28 rotor),followed by resuspending and incubating at 37 EC for 2 hours in 1/200 ofstarting volume of Tris-buffered saline (TBS, pH 7.8) containing 10 mMMgCl₂ dNTPs (0.1 mM each), 3 mM spermine, and 0.3 mM spermidine. After asecond ultracentrifugation at 50,000 g for 30 minutes (Beckman TLA-100.3rotor), the vector pellet was resuspended in 1/2000 of the initialvolume of TBS with 2 :g/ml Polybrene and was stored at −80 EC.

Titration assay for vector potency. Human 293 embryonic kidney cells ormurine NIH 3T3 cells were subcultured into six-well culture plates(Beckton Dickinson, Franklin Lakes, N.J.) at 10⁵ cells/well with DMEMcontaining 10% FBS (Life Technologies Inc.). After 8 hours had passed,cells were exposed to serial dilution of vector stocks in the presenceof Polybrene (8 :g/ml). Titers were scored 48 hours after transductionby FACS analysis to quantitate GFP-expressing cells. All assays weredone in triplicate.

In vivo vector administration to mice. Thirty-one 8 week-old normalBALB/c mice (15 male, 16 female) were obtained from Charles RiverLaboratories (Wilmington, Mass.) and housed in a pathogen-free facilityon a 12-hour light/dark cycle. One week later, the mice were randomlydivided into control (7 male, 7 female) and treated groups (8 male, 9female). The treated groups were injected i.v. with 100 :l pooledconcentrated vector stock into the tail vein over 3-6 seconds; thecontrol groups were injected with 100 :l TBS containing 2 :g/mlPolybrene. All animal procedures were done under aseptic conditions andin accordance with protocols approved by the Institutional Animal Careand Use Committee. Mice were periodically bled by the retro-orbitaltechnique.

Perfusion and organ collection. At either 4 or 40 days after injection,the mice were euthanized by intraperitoneal administration of anoverdose of sodium nembutal (Abbott Laboratories). Each mouse wasperfused transcardially through the aorta with PBS for 5-10 minutesuntil its liver turned pale, followed by perfusion with 4%formaldehyde-PBS for 10-15 minutes. Nine organs were removed in thefollowing order: gonad, bladder, gastrointestinal tract, lung, heart,kidney, liver, spleen, and brain. Organs were post-fixed in 4%formaldehyde-PBS for 0.5 to 2 hours, and then transferred into a 30%sucrose-PBS solution for storage at 4 EC until further processing. Bonemarrow was harvested in PBS from femur and tibia of each mouse, andstored in Cell Lysis Solution (Puregene, Minneapolis, Minn.) for furtherDNA isolation. The remains of mice were stored in 4% formaldehyde-PBSfor pathologic analysis. Aliquots of each organ were segregated for twodifferent assays. Genomic DNA was isolated from the first aliquot usinga DNA isolation kit (Puregene) for quantitation of GFP gene. The secondaliquot was immersed in 10% neutral-buffered formalin for 1-3 days andembedded in paraffin by routine methods. Sections of 4 to 6 :m, stainedwith H&E, were examined.

Real-time QPCR using concurrent reactions. Both GFP transgene andendogenous mouse Apob sequence (as an internal control) were quantitatedsimultaneously in the same reaction well of a total 50 :l PCR volume byreal-time PCR. The TaqMan probe for detection of GFP transgene waslabeled with fluorescent reporter dye 6FAM at the 5N end and quencherdye TAMRA at the

3Nend (5N-CCGACAAGCAGAAGAACGGCATCA-3N;, SEQ ID NO: 1)whereas the probe for Apob was labeled with fluorescent reporter dye VICat the 5N end and quencher dye TAMRA at the 3N end(5N-CCTTGAGCAGTGCCCGACCATTC-3N; SEQ ID NO:2). Sequences of TaqManprimer-probe sets for GFP (sense, 5N-ACTACAACAGCCACAACGTCTATATCA-3N; SEQID NO:3; antisense, 5N-GGCGGATCTTGAAGTTCACC-3N; SEQ ID NO:4) and Apob(sense, 5N-CGTGGGCTCCAGCATTCTA-3N; SEQ ID NO:5; antisense,5N-TCACCAGTCATTTCTGCCTTTG-3N; SEQ ID NO:6) were designed using thePrimer Express program (PE Applied Biosystems, Foster City, Calif.). Theduplex reaction contained 0.01-1 :g genomic DNA, 200 nM of each GFPprimer, 200 nM GFP probe, 40 nM of each Apob primer, 200 nM Apob probe,and 25 :l TaqMan 2 H Universal Master Mix (PE Applied Biosystems)including 8% glycerol, 1 HTaqMan buffer A, 5 mM MgCl₂, 400:M dUTP, 200:M dATP, dCTP, AND dGTP (each), AmpliTaq Gold (0.025 U/:l), and AmpEraseUNG (0.01 U/:l). All PCR reactions were set up in a MicroAmp Optical 96well Reaction Plate (PE Applied Biosystems). Amplification conditionswere 2 minutes at 50 EC and 10 minutes at 95 EC for the first cycle,followed by 50 cycles of 95 EC for 15 seconds and 60 EC for 1 minute.The TaqMan probes were cleaved during amplification, generating specificfluorescence emission for the FAM-labeled GFP probe or the VIC-labeledApob probe. The data were collected in real time from the ABI PRIME 7700Sequence Detector and transferred online to a Macintosh 7100 foranalysis using the Sequence Detector version 1.6 program (PE AppliedBiosystems). Unknown samples were run in triplicate, and standardsamples were in duplicate. All threshold cycle (Ct) values of GFPtransgene were normalized by Ct of Apob that measured the total DNAcontent in each individual reaction. Transgene frequencies of unknownsamples were interpolated from a standard curve (ranging from 0.001% to100%) that was established by simultaneous amplification of a series ofgenomic DNA mixtures derived from a mouse myeloid cell line (32Dp210)(Carlesso et al., 1994) and a GFP-containing cell line(32Dp210-LNChRGFP) with 1 copy per genome (as determined by Southernblot analysis).

Flow cytometry. Blood samples were collected using heparinizedhematocrit tubes and diluted 1:1 into heparin sodium solution (ICNBiomedicals Inc., Aurora, Ohio). PBL were fixed in Optilyse B solution(Immunotech, Marseille, France), while red cells were further lysed bythe addition of dH₂O. Specific subsets of mouse PBL were detected bystaining with PE-conjugated hamster monoclonal antibody to mouse CD3efor T lymphocytes (Pharmingen, San Diego, Calif.), or PE-conjugated ratmonoclonal antibody to mouse CD45R/B220 for B lymphocytes (PharMingen).PE-conjugated Armenian hamster immunoglobulin group 1 and ratimmunoglobulin G2a were used, respectively, as isotype controls.Cultured cells were trypsinized, then fixed in 4% formaldehyde, anddiluted in PBS to a concentration of 5 H 10⁵ cells/ml. Two-color FACSanalysis for cellular GFP (FL-1) and PE staining (FL-2) were carried outby FACSCalibur with the CellQuest program (Becton Dickinson). Cells frommice injected with TBS were analyzed as negative controls for GFPexpression in blood samples. For titration assays, human 293 cells ormouse 3T3 cells were used as negative controls; whereas uninducedSODk1-CGFI packaging cells were used as positive controls.

Immunohistochemical staining for GFP-expressing cells. After several PBSrinses and an incubation in 3% hydrogen peroxide, the fixed cryosectionswere blocked in 5% normal goat serum (Vector Labs, Burlingame, Calif.).The sections were then incubated with the primary anti-GFP antibody(1:50; Clontech Lab) in 5% goat serum-0.1% Triton X-100 overnight at 4EC. After rinsing, the sections were incubated in the biotinylatedrabbit anti-goat secondary antibody (Vector Labs) for 1 hour, washedthree times with PBS, stained with biotinylated horseradishperoxidase-avidin (ABC kit; Vector Labs), and then colorized by using adiaminobenzidine (DAB) substrate kit (Vector Labs). After staining,sections were washed in dH₂O, air-dried, and mounted in Permount (FisherScientific Co., Fairlawn, N.J.).

Results Vector Concentration and Administration

To assess the potency of the first-generation lentiviral vectorHRNcmvGFP, vector stocks from several batches of inductive productionand concentration were thawed at 30 EC and pooled. Transduction wasevaluated with both human 293 cells and mouse NIH 3T3 cells by FACSanalysis for GFP expression (Table 1). Up to 780-fold increase in titerwas observed in concentrated vector supernatants, resulting in 1.8∀0.15H 10⁸ transforming units (TU) per milliliter. Titers obtained from human293 cells were always about 10-fold higher than those from mouse NIH 3T3cells. Thus, about 2H 10⁷ 293 TU of recombinant HIV-GFP (in 100 :l) wasinjected through the tail vein into each of 17 normal BALB/c mice.

TABLE 1 Potency of lentiviral vector generated from packaging cell lineTiter* determined by FACS analysis for GFP expression (TU/ml) Beforeconcentration After concentration Cells Mean SD Mean SD Human 293 2.3 H10⁵ ∀ 0.87 H 10⁵ 1.8 H 10⁸ ∀ 0.15 H 10⁸ Murine NIH 2.6 H 10⁴ ∀ 0.12 H10⁴ 1.2 H 10⁷ ∀ 0.76 H 10⁷ 3T3 *Vector-containing supernatants werestored at −70EC and thawed in 30EC water-bath before titration assay.

Pathology

To evaluate toxicity due to the i.v. administration of VSVG-pseudotypedlentiviral vector, hematoxylin and eosin (H&E)-stained sections from 17treated and 12 control mice were examined by light microscopy (Table 2).Mucosal and submucosal edema was found in the gastrointestinal tract of14 treated and 6 control mice, and congestion was observed in the liversof both control and treated mice. These changes appear to have resultedfrom the perfusion and fixation procedure. One mouse (F11) developed abrain abscess, presumed to be the result of injury from the bloodcollection procedure. Lymphocyte infiltration or other signs ofinflammation were not observed in any of the examined organs. Thus, nosignificant lesions attributable to the test article were found in anyof the tissues.

TABLE 2 Pathological findings of treated and control animals MouseNumber Liver Spleen Lung Heart Kidney Brain GI tract Gonad Skin* 4 Dayspost-administration Control ConM1 NSL NSL NSL NSL NSL NSL — NSL NSLConM3 NSL — NSL NSL NSL NSL edema NSL — ConF4 Cong NSL — — — — edema —NSL ConF5 — — NSL NSL NSL NSL NSL NSL — ConF6 NSL NSL NSL NSL NSL NSLedema NSL NSL Treated TrM12 NSL NSL NSL NSL NSL NSL edema — — TrM13 NSLNSL NSL NSL NSL NSL NSL NSL — TrM14 NSL NSL NSL NSL NSL NSL edema NSLNSL TrF13 NSL NSL NSL NSL NSL NSL edema NSL — TrF14 NSL NSL NSL NSL NSLNSL edema NSL NSL TrF15 NSL NSL NSL NSL NSL NSL edema NSL NSL 40 Dayspost-administration Control ConM5 Cong NSL Hemo NSL NSL NSL edema — NSLConM6 NSL NSL NSL NSL NSL NSL NSL NSL — ConM15 NSL NSL NSL NSL NSL NSLedema NSL — ConF1 NSL NSL NSL NSL NSL NSL edema — NSL ConF2 NSL NSL NSLNSL NSL NSL NSL NSL NSL ConF3 NSL NSL NSL NSL NSL NSL NSL — — ConF16 NSLNSL NSL NSL NSL NSL NSL NSL — Treated TrM7 Cong NSL NSL NSL NSL NSLedema NSL — TrM8 Cong NSL NSL NSL NSL NSL edema NSL — TrM9 NSL NSL NSLNSL NSL NSL edema NSL — TrM10 Cong NSL NSL NSL NSL NSL edema — NSL TrM11NSL NSL NSL NSL NSL NSL edema NSL — TrF7 NSL NSL NSL NSL NSL NSL NSL —NSL TrF8 NSL NSL NSL NSL NSL NSL edema NSL — TrF9 Cong NSL NSL NSL — NSLedema — NSL TrF10 NSL NSL NSL — NSL NSL edema NSL — TrF11 NSL NSL NSLNSL NSL abscess NSL edema — TrF12 Cong NSL — — — — edema — — NSL, nosignificant lesions were found. Cong, small foci of sinusoidalcongestion. Hemo, small foci of extravasation of blood. Edema, mucosaland submucosal edema due to perfusion and fixation procedures. —, Notexamined. *Tail was examined at the injection site.

Duplex Real-Time PCR for GFP and Apob

To achieve a high degree of sensitivity, reproducibility, and accuracyin quantitating gene transfer efficiency, a real-time QPCR assay wasestablished for concurrent quantification of GFP and endogenous mouseapolipoprotein B (Apob) in a single reaction vessel. The amplificationplots for GFP shift to the right as initial transgene input is reduced(from 100% to 0.001%) while same-well Apob amplifications remainconstant because total DNA content is similar in all samples (that is, 1:g/well as determined by spectrophotometry). A common threshold wasselected in the exponential phase of PCR reactions to determine aspecific threshold cycle number for each sample.

To determine whether the concurrent amplification of GFP and Apob in thesame well was occurring under optimized reaction conditions,amplifications of GFP were compared with same well Apob from a serialdilution (>5-log-fold) of a reference cell preparation, that is, cellscontaining one copy of GFP per cell. Theoretically, if the reactionefficiencies for both genes were the same and constant across variousconcentrations of DNA template, the difference in amplifications betweenthe two targets should remain constant. This proved to be true, asindicated by the two parallel amplification curves for GFP and Apob, andfurther confirmed by graphing the difference in threshold cycle numbers( )Ct) against the log of the DNA dilution fold. Regression analysis ofthese data demonstrated a horizontal line with a negligible slope(0.04). This result indicated that the PCR efficiency for GFP wascomparable to that for Apob in this duplex reaction system, thusconfirming that concurrent amplification of Apob would serve as areliable internal reference against which to normalize GFP measurements.

To evaluate further the sensitivity, accuracy, and reproducibility ofthis real-time QPCR assay, standard curves were established by plottingnormalized threshold cycle number (nor CT) against transgene frequencyusing a set of standard samples. Although each of the standardscontained a different percentage of GFP transgene, the totalconcentration of DNA remained similar. When Ct was normalized for DNAconcentration on the basis of equal optical density (OD) readings usinga spectrophotometer, the linear regression analysis of the curveindicated a slope of −4.02, with r² of 0.984. An even higher squaredcorrelation coefficient (0.999) was observed in standard curve using thesame set of samples when normalizing Ct-GFP with in-well Apob readings.These results indicate an extremely efficient quantitation assay thatremains linear (5-log fold) from 100% to 0.001% transduced cells.Considering that 1 :g DNA is equivalent to about 10⁵ cells, this assaycan detect as few as one copy of GFP per reaction. Moreover, there was ahigh reproducibility of this real-time QPCR. A “mean standard curve” wasgenerated by analyzing nor Ct values derived from 20 PCR reactions(amplified in 10 separate runs) of the same set of samples. The standarddeviation was <3% of nor Ct for each of the standard samples, suggestinga stable high reproducibility over the 5-log-fold quantitation.

Biodistribution of GFP Transgene 4 Days after Administration

To assess where, and to what degree, the HIV-based GFP-containinglentiviral vector localized within the animals shortly after vectoradministration, 10 different organs were carefully collected from sixtreated and six control mice 4 days after injection. Mice were perfusedtranscardially for 20 minutes to minimize potential DNA contaminationfrom blood. Cross sections of each organ were examined for GFP transgeneby real-time QPCR assay. High levels of GFP were observed in bone marrow(ranging from 5 to 37 GFP copies per 100 genome equivalents), liver(12-59), and spleen (20-54) in treated mice. In contrast, transgene wasundetectable in the brain of one animal and the gastrointestinal tractof all treated mice. Various amounts of transgene were obtained in otherorgans from all treated animals, ranging from 0.01 to 1 GFP copy per 100genome equivalents. These observations demonstrated a variabledistribution of in vivo-transduction capability of lentiviral vectors.

Biodistribution of GFP Transgene 40 Days after Administration

To assess stably transduced transgene efficiency in treated mice, organdistribution of GFP was determined in mice 40 days after injection(Table 3). Relatively high levels of GFP were observed in liver (rangingfrom 0.3 to 1.3%) and spleen (ranging from 0.045 to 0.38%), which weresubstantially lower than those in mice 4 days after injection. Gonadsfrom all but one treated animal (TrF9) contained undetectable to barelydetectable levels of GFP (<9 copies GFP per 10⁵ cells). Fromundetectable to 0.30% transgene was found in other organs. Remarkably,very high levels of transgene (4.7-22.7%) were observed in bone marrowfrom all but one mouse. These levels are comparable to those observed inmice 4 days after treatment. These results suggested that bone marrowmight be the organ most accessible to VSVG-pseudotyped lentiviralvector.

TABLE 3 Tissue distribution of GFP in mice 40 days after vectorinjection (%) Mouse Number Gonad Bladder GI tract Brain Kidney HeartLung Spleen Liver BM Male Control ConM4 UD 0.027 UD na na UD UD UD UD UDConM6 UD UD UD UD UD UD UD UD 0.02 UD ConM15 UD UD UD 0.003 UD 0.002 UDUD UD na Treated TrM7 0.005 0.037 UD 0.061 UD na UD 0.267 0.639 17.94TrM8 0.008 0.087 0.002 0.161 0.002 0.043 UD 0.137 0.550 na TrM9 0.0030.060 0.004 UD 0.001 0.015 UD 0.235 0.468 13.56 TrM10 0.003 0.022 UD0.007 0.002 0.023 0.003 0.202 0.762 na TrM11 UD 0.040 UD 0.023 0.0030.077 0.304 0.045 1.263 14.54 Female Control ConF3 UD UD UD UD UD na0.005 UD UD UD ConF4 0.003 na UD 0.003 UD UD 0.001 UD UD na ConF16 UD UDUD UD UD UD UD UD 0.01 UD Treated TrF7 0.006 na UD 0.005 UD 0.003 0.0730.289 0.256 na TrF8 0.006 0.066 UD UD UD 0.001 0.002 0.183 0.713  4.743TrF9 0.034 0.058 UD 0.053 0.003 0.007 0.008 0.166 0.878  7.895 TrF10 UD0.202 UD 0.014 0.02 UD 0.060 0.072 1.292 15.12 TrF11 0.009 UD 0.0010.111 0.001 0.022 0.026 0.132 0.969  0.208 TrF12 na 0.846 UD 0.102 0.003UD 0.003 0.377 0.728 22.68 UD, un-detectable. na, not available.

Transgene Frequency in Peripheral Blood Leukocytes

To monitor the transgene in blood cells, whole blood was collectedperiodically on days 4, 11, 25, and 40 after injection and analyzed byreal-time QPCR. GFP transgene frequency in peripheral blood leukocytes(PBL) decreased significantly (P<0.0001) from a mean of 0.77% (∀0.27) onday 4 to 0.07% (∀0.04) on day 11. Interestingly, the GFP level increasedback to 0.73% (∀0.47) on day 25, a comparable result to that on day 4(P=0.858). Remarkably, it continued to increase to a level of 20.8%(∀17.1) on day 40 following injection (P<0.002), indicating the presenceof an additional source for GFP⁺ leukocytes. This latter result isconsistent with the earlier observation of high levels of GFP transgenein bone marrow from mice assayed 40 days after injection.

Transgene Expression in PBL by FACS Analysis

To determine whether the genetically translated GFP is biologicallyactive, GFP expression and the murine B-cell or T-cell subsets wereanalyzed by two-color flow cytometry with phycoerythrin (PE) conjugatesin whole blood collected periodically. FACS analysis failed to identifyGFP-expressing cells in PBL on days 4, 11, and 25, although low levelsof GFP transgene were detected by real-time PCR (<1%). However, up toabout 10% of leukocytes were found to be GFP in mouse TrM7, whose bloodcontained the highest level of transgene (62.7%) 40 days afterinjection. Moreover, GFP-expressing cells included both CD45R/B220⁺ Bcells and CD3e⁺ T cells.

Transgene Expression in Liver Visualized by Immunochemical Staining

To characterize transduced cell type(s) further, mouse livers werestudied that had relatively high levels of gene transfer by QPCR byimmunochemical staining for GFP cells. In livers 4 days after injection(with 12-59% transgene frequency), the GFP cells (reddish-brown stainingin cytoplasm) were most likely to be hepatocytes adjacent to bloodvessels. However, no identifiable GFP⁺ cells were detected in liverderived from mice 40 days after injection; approximately 1% transgenefrequency was measured by real-time QPCR (Table 3).

Discussion

With >300 phase I/II clinical trials conducted worldwide over the lastdecade, gene therapy represents one of the fastest growing areas inexperimental medicine (Romano et al., 2000). Tissue biodistribution isan important aspect of characterizing new vectors, one that has receivedgreat attention from the FDA and the National Institute of Health's(NIH) Recombinant Advisory Committee (FDA, 1991; FDA, 1998; Pilaro etal., 1999). However, only limited data are available from preclinicaleffectiveness and toxicity studies needed to evaluate these new products(Verdler et al., 1999). HIV-based lentiviral vectors are promising toolsfor in vivo gene therapy, but their safety issues are more criticalbecause of their origins. Although gene transfer and transgeneexpression of VSVG-pseudotyped HIV-based vectors have been demonstratedby several groups in various organs (Naldini et al., 1996; Kafri et al.,1997; Johnson et al., 2000; Miyoshi et al., 1997; Woods et al., 2001),the biodistribution and systemic effects have not been assessed. In thisstudy, the organ biodistribution and general toxicity of afirst-generation lentiviral vector after tail-vein injection in mice wasassessed. A real-time QPCR assay was established with a broad range ofquantitation (5-log fold) to detect as few as one copy of GFP per 10⁵cell genomes. Such studies are crucial in understanding both thepotential efficacies, as well as the level of risk for germlinetransmission (Pilaro et al., 1999). The unexpected observation of highbone marrow transgene frequency (ranging from 0.21 to 22.7% of totalbone-marrow genome) has important implications for stem cell genetherapy.

Accurate quantitation of gene transfer (or gene correction) has been auniversal challenge to the field of gene therapy. High sensitivity andreproducibility of such assays are key requirements for a successfulbiodistribution study, especially when extremely low gene transfer isanticipated in some of the nontarget organs such as gonad (Gordon,1998). PCR-based DNA analysis has been specified as an adequate methodin biodistribution studies by the FDA (Pilaro et al., 1999). A majorimpediment to the use of PCR as a quantitative technique has been theinherent change in kinetics of the chemical reaction over time assubstrates and other essential components are consumed (Orlando et al.,1998). With a given PCR cycle number (for example, 25 cycles), PCRamplifications from different amounts of initial target templates couldbe at different stages (geometric, linear, and plateau phases) withdivergent amplification rates and efficiencies. Therefore, conventionalend-point PCR methods are limited by the sensitivity of detection inboth linear and plateau phases of the PCR. However, a systemicbiodistribution study of gene transfer frequency demands the capabilityto quantitate target templates over several-log fold.

A new technique for quantitating PCR products in real time has beendeveloped recently (Held et al., 1996; Becker et al., 1999). It is ableto identify and measure amplification signals in “real time” (that is,every 7 seconds) during PCR reactions. The real-time QPCR assayestablished here could measure as few as one copy of target sequence ina background of about 10⁵ genomes (1 :g DNA). Moreover, reproducibilityand accuracy were consistently high over a wide range of quantitation,with 5-log-fold difference in the amount of target templates (1 to 10⁵copies). In addition, the assay was further optimized by simultaneouslyquantitating both target GFP sequence and internal-control Apob sequencein the same reaction. In each reaction well, the amplification of Apobserves as an internal reference to validate each reaction mixture (anextremely important issue when the target sequence is very low orundetectable). Such a modification also has the advantage of reducingsampling and other systemic errors, and eliminates the need for a secondset of control reactions. Thus, it is more accurate and economical.

Wild-type vesicular stomatitis virus (VSV) has a broad host rangeextending from insects to nearly all mammals (Schnitzlein et al., 1985).In humans, VSV infections result in nonsevere influenza-like symptoms(Fields et al., 1967). The VSV glycoprotein G is the major antigenicdeterminant responsible for virus attachment and membrane fusion (Coll,1995). Unlike most viral envelope proteins, which must bind to aspecific cell-surface protein receptor to mediate infection (Albrittenet al., 1989; Sattentau et al., 1986), VSVG interacts with an intrinsicphospholipid component of the plasma membrane (Mastromanno et al., 1987;Knoieczko et al., 1994). Moreover, the VSVG also has the unique abilityto withstand the shearing forces encountered during ultracentrifugation.Therefore, to broaden the target cell range and increase potency invector preparations, VSVG has been utilized to “pseudotype” gene therapyvectors such as retroviral (Burns et al., 1993), HIV-1-based (Naldini etal., 1996), and FIV-based (Poeschla et al., 1998) lentiviral vectors.

In vitro studies on VSVG-pseudotyped HIV-1-based vectors havedemonstrated efficient gene transfer into nondividing airway epithelialcells (Goldman et al., 1997), unstimulated primary T lymphocytes(Costello et al., 2000), non-prestimulated CD34⁺ cells (Douglas et al.,1999), terminally differentiated macrophages, and peripheral bloodmonocyte-derived dendritic cells (Schroers et al., 2000). By route oflocal administration, in vivo gene delivery has been accomplished in ratbrain (Naldini et al., 1996; Blomer et al., 1997), in liver and muscle(Kafri et al., 1997), in retina (Mixoshi et al., 1997), and in airwayepithelia (Johnson et al., 2000). In this study, overall in vivo organdistribution was assessed by injecting a low dose of lentivirus i.v.into mice (2 H 10⁷ IU/mouse). Relatively high gene transfer was observedin bone marrow (ranging from 0.21 to 22.7% transgene frequency), liver(0.26-1.3%), and spleen (0.045-0.38%) from mice 40 days after injection.Variable low levels of transgene were observed in bladder (fromundetectable to 0.85%), lung (from undetectable to 0.30%), heart (fromundetectable to 0.021%), brain (from undetectable to 0.16%), kidney(from undetectable to 0.003%), and gastrointestinal tract (fromundetectable to 0.004%). These observations do not conflict with thoseobserved by others (Park et al., 2000), in which a relatively high doseof lacZ-containing lentivirus (1 H 10⁸ TU) was injected into the portalvein of mice. The expression of 3-galactosidase was detected by X-galstaining in liver (0.16∀0.08% of hepatocytes) and spleen, but not in thebrain, heart, lung, kidney, and duodenum.

The GFP transgene frequency was surprisingly high in liver (up to 59%)and spleen (up to 54%) from mice 4 days after injection, and decreaseddramatically to a maximum of only 1.3% in liver and 0.38% in spleen frommice 40 days after injection. This change may be due to the existence ofabundant defective vector particles that contain partial reversetranscripts, and the loss of extrachromosomal proviral DNA. It has beenreported that only 0.1-1% of the virus particles in VSVG-pseudotypedlentiviral vector preparations were infectious, when using the minusstrong-stop cDNA fragment that was present in viral capsids as templatefor real-time QPCR (Scherr et al., 2001). Therefore, in this study about2 H 10¹¹ particles were injected into each mouse, with about 2 H 10⁷transduction units. Varied partial reverse-transcription (RT)intermediates have been found to be present in newly assembled HIV-1particles (Trono, 1992). These cDNA intermediates and their derivativesmay have contributed to the GFP transgene signal in 4-day animals.Moreover, it has been found that unintegrated lentiviral proviral DNAmay persist in transduced TE671 (muscle), 293T (kidney), and HepG2(liver) cells for more than 4-5 passages, but disappear by 40 passages(Chang et al., 1999). Transient GFP expression caused byintegrase-defective lentiviral vectors was observed for as much as 10days in CD34⁺ cells and 14 days in 293 cells (Haas et al., 2000). Nolymphocyte infiltration or other signs of inflammation were observed inany liver or spleen samples from mice 4 days or 40 days after injectionin this study, although a transient elevation was observed in serumalanine amino-transferase level by others (Park et al., 2000). Thus, theloss of GFP transgene in the liver is unlikely to be related to the lossof transduced cells resulting from liver toxicity.

One of the most disturbing concerns for gene therapy in humans is thepossibility of germ line integration of transgene, which might result inthe introduction of heritable genetic changes into the offspring ofpatients (Pilling, 1999). Germline integration may lead to insertionalmutations that might have devastating consequences, as indicated in someof the transgenic mice produced by pronuclear microinjection (Woychik etal., 1985; McNeish et al., 1988). To assess the risk of germlineintegration by a first-generation lentiviral vector, transgene frequencywas quantitated in whole gonads of mature mice (Table 3). Fromundetectable to 9 copies/10⁵ genome levels of transgene were found inall testes (n=5), and all but one ovary (n=5). A very low level oftransgene (3 copies/10⁵ genomes) was observed in one of the six controlanimals, although special precautions were taken during perfusion,necropsy, DNA isolation, and QPCR assay. Thus, the possibility ofcross-contamination cannot be ruled out. In addition, very high levelsof transgene (2.08∀1.7 H 10⁴ copies/10⁵ genomes) detected in the PBL oftreated mice may contaminate the gonad if not eliminated completely byperfusion. Even if the transgene found in the gonads is real, thehematopoietic spread of lentiviral vector to spermatocytes or developingoocytes is unlikely because they are relatively inaccessible to largemolecules or to infection by viruses (Gordon, 1998). Moreover, there arestatistical considerations that mitigate against the germline transferof foreign DNA that reaches an offspring. For example, integration oftransgene into one spermatogenic cell would lead to the genetictransformation of only a few of the millions of cells that wouldultimately reach the ejaculate. Also, of the about 400,000 oocytespresent in the human ovary at the onset of menstruation, only a fewhundred are ovulated during the reproductive lifespan of a woman, andfewer than a dozen of those ovulated oocytes contribute their genes tosubsequent offspring. Thus, together with the observations providedherein, the risk of germline transmission of the first-generationlentiviral vector by i.v. administration is very low.

The most surprising observation was that bone marrow exhibited thehighest transgene frequency (ranging from 0.21 to 22.7% of totalbone-marrow genome) in all mice tested 40 days after injection (Table3). This was consistent with the observation that high levels oftransgene were detected in PBL from these animals (ranging from 0.61 to62.7%). It was also supported by the results that up to 10% of PBLexpressed GFP as determined by FACS analysis. In addition, the transgenelevels in PBL decreased significantly from a mean of 0.77% on day 4 to0.07% on day 11, and then increased considerably back to 0.73% on day 25and to 20.8% on day 40 following injection. This observation suggestedthe presence of an additional resource for GFP⁺ leukocytes, implying thetransduction of hematopoietic progenitor cells. This study provides thefirst indication that bone marrow may be a susceptible target tissue fori.v. administration of VSVG-pseudotyped lentiviral vectors. It ispossible that intravenously delivered lentiviral vector may reach stemcells; ex vivo transduction studies have demonstrated the capability oflentiviral vectors to transduce more primitive and quiescent stem cells(Woods, 2001; Case et al., 1999). The i.v. approach may overcome some ofthe difficulties encountered by ex vivo approaches, such as limited genetransfer efficiency, maintenance of long-term engraftment of thetransduced cells, and in vitro manipulation steps (Richter et al.,2001).

Example II

Mucopolysaccharidosis type I (MPS I) is an inborn error of lysosomalglycosaminoglycan (GAG) metabolism resulting from deficiency ofalpha-L-iduronidase (IDUA). While allogeneic hematopoietic stem cell(HSC) transplantation results in systemic metabolic correction,including prevention of neurologic damage, attempts to exploit Moloneymurine leukemia virus vectors have failed to demonstrate the requisitequalities to merit substitution of ex vivo HSC gene therapy forallogenic HSC transplantation. Lentiviral vectors may integrate intonon-dividing cells and may have broad tropism when pseudotyped withVSV-G envelope, thus potentially solving this problem.

A murine model of MPS I (kindly provided by Hong-Hua Li and Elizabeth F.Neufeld; Zheng et al., 2001) was used to evaluate transgene expressionin fibroblasts from these mice. Mice were genotyped with a SYBR Greenassay from which primary skin fibroblast cultures were established.Second- and third-generation HIV-1 based vectors were generated totransduce these IDUA-deficient murine fibroblasts with either GFP orIDUA. Second generation (three-plasmid) and third generation(four-plasmid) SIN lentiviral vectors were prepared with either GFP(pCS-CG) or IDUA (pCS-P1) transgenes by co-transfection into 293T cells.Potency of 48 hour viral supernatants was assessed by two methods. Inthe first assay, real-time quantitative PCR was exploited using aprimer-probe set for the minus strong-stop cDNA (U5/R region) ofencapsulated lentiviral genomes (Scherr et al., 2001) yielding titersbetween 2×10⁶ genomes/mL and 5×10⁷ genomes/ml. In the second assay, 293Tcells were exposed to GFP vector supernatants, and then subjected toFACS analysis to select for transduced cells yielding titers of 1×10⁵TU/mL. Third-generation vectors were found to be comparable tosecond-generation preparations.

Murine IDUA-deficient fibroblasts (2.5×10⁵ cells/plate) were exposed tovarious concentrations of vector and incubated for 24 hours beforechanging the media, and then cultured for an additional 6 days prior toanalysis. Cells transduced with third generation IDUA (pCS-P1) werefound to have markedly increased IDUA enzymatic activity (60-120nmol/mg/hr) comparable to that of normal human fibroblasts orleukocytes. Quantitative PCR assays for the IDUA and GFP transgenesquantified the level of integration of the transgene within the genome(see Example I). The real-time QPCR assays for the third generation IDUAand GFP vectors found that the average percentage of transduced cellswas 60% whereas the negative control was 0%. Microscopy analysis of GFPtransduced cells showed similar results.

For in vivo transduction, a transgene plasmid (pCS-P1) containing thehuman IDUA cDNA sequence under transcriptional control of the human PGKpromoter was prepared. Vector preparations were generated by transientcotransfection of 293T cells using a third-generation 4 plasmidpackaging system with Rev function (pEFRev) separated from other helperfunctions (p2NRF) (kindly provided by T. Kafri et al. Real-timequantitative PCR methods were exploited to determine lentiviral particlenumbers in vector preparations, and to assess transduction efficiency(Example I).

Intravenous injection of an IDUA encoding replication-defective (thirdgeneration) VSVG pseudotyped lentiviral vector (e.g., 5×10⁷ TU; FIG. 1)into newborn mice resulted in higher-than-normal levels ofalpha-L-iduronidase in blood (FIG. 2). The circulating levels were up to1-log-fold higher than normal circulating levels, and those levels weremuch higher than those achieved in efficacious bone marrowtransplantation. Further, the circulating levels were up to at least1-log-fold higher than those achieved by any other means of genetherapy, e.g., intravenous injection of a retrovirus vector in a mousemodel of mucopolysaccharidosis type VII (Xu et al., 2002). Moreover,circulating levels of enzyme persisted throughout the period ofobservation (i.e., 3 months after treatment). Because lentiviral vectorsintegrate the therapeutic gene into the host chromosome, therapeuticlevels of protein (enzyme) are expected to persist for long periods oftime, probably for the lifetime of the treated individual.

In addition, intravenous infusion of newborn MPS I mice with an IDUAencoding recombinant lentivirus resulted in normal facial appearance(FIG. 3) indicating the efficacious effect of transgene expression onbone growth. Further, intravenous infusion of newborn MPS I mice withthe IDUA encoding vector also resulted in normal parameters of behavior(FIG. 4) indicating the efficacy of this treatment on the progressivebrain degeneration of MPS I. Moreover, FIG. 5 shows a micrograph of thepathology observed in mice with Hurler syndrome as well a micrograph ofIDUA lentivirus treated mice which demonstrates that treated mice have adecrease or lack of GM-2 ganglioside pathology.

Example III Materials and Methods

Plasmids. The plasmid for 5B transposon expression, pT-CAGGS-GUS(transposon), was constructed as follows. A 2.3 kb fragment containinghuman GUSB cDNA was excised from pHUG13 (ATCC 95658) by EcoRI digestionand ligated to EcoRI sites in the poly linker in pCAGGS. The resultingexpression cassette for GUSB expression included a cytomegalovirusenhancer, chicken β-actin promoter, the initial intron of the chickenβ-actin gene, GUSB cDNA, and a rabbit β-globin and SV40 polyadenylationsignal. This expression cassette was inserted between SspI and HindIIIsites of the pT/BH transposon polylinker. The SB transposase expressionplasmid pCMV-SB10 has been previously described (Ivics, 1997).Pyrogen-free plasmids were used in this study and were isolated usingQiaFree kit (Qiagen, City, State).

Mice. MPS VII mutant mice (B6.C-H-2bml/ByBir-gus^(mps)) were obtainedfrom Jackson Laboratories (Bar Harbor, Me.) and maintained in theAAALAC-accredited Specific Pathogen-Free mouse facility at theUniversity of Minnesota. Homozygous mutant mice were produced bybreeding of heterozygotes. Genotyping was performed by allelicdiscrimination assay using TaqMan chemistry.

Injections. The plasmids were injected into the tail vein using a 3-cclatex-free syringe with a 271/2 G needle. The hydrodynamics-basedprocedure was performed as described in Wolff et al. (2000). Each mousereceived 25-37.5 μg of plasmid DNA in lactated Ringers solution, in atotal volume equal to 10% of body weight. One animal from each groupdied before completion of the experiment: one mouse from Treatment Group1 and one mouse from Treatment Group 3 died on 1 week post-injection andone mouse from Treatment Group 2 died 4 weeks post-injection. The organsfrom these mice were resected within 8 hours of death and used forenzyme quantification and GUSB histochemical staining.

Treatment regimens. In the short-term experiment, four groups of MPS VIIor wt mice (n=4 each) received 25 μg of pT/BH-CAGGS-GUS (MPSVII andtreatment groups) or pBluescript (MPS VII and wt control groups). Themice were euthanized by CO inhalation 48 hours after injections. 400 μlblood was drawn from the heart for plasma isolation, and livers wereextracted and preserved for biochemical molecular, histochemical andpathological analysis.

For the long-experiment, MPS VII mice age 4-26 weeks were used. Analiquot (25 μg) of a single preparation of transposon pT-CAGG5-GUSB wasinjected either alone (Treatment Group 1), or with pCMV-SB10 at 1:1(Treatment Group 2) or 10:1 (Treatment Group 3) molar ratios. The amountof injected DNA was kept the same in each group (37.5 μg) with thefiller plasmid, pBluescript. The control group of MPS VII mice wasinjected with the filler plasmid alone. All injections were performedonly once.

The mice were bled by retroorbital phlebotomy 48 hours, 1 week, 2 weeks,and 4 weeks post-injection to obtain plasma for enzyme assays. 8 weekspost-injection, mice were euthanized by CO₂ inhalation, and the organs(liver, spleen, heart kidney, lung, brain, and gonads) were resected,cut into 2 mm³ sections and preserved in different ways for analysis.Prior to organ resection, 400 ml of blood was drawn for plasma and whiteblood cell (WBC) isolation.

For lysosomal enzyme quantification, plasma and tissues were snap-frozenin liquid nitrogen and stored at −80° C. Activities of GUSB,alpha-galactosidase and total β-hexosaminidase were measured in tissuehomogenates and plasma using fluorometric assay. Protein concentrationswere determined with Bradford assay (Bradford, 1976) using Bio-Radreagent.

For histology and histopathology studies, tissues were fixed in 10%neutral-buffered formalin, embedded in paraffin and sectioned at 6 μmfor staining with Hematoxylin and Eosin.

Histochemical localization of GUSB was performed in 6-8 μm frozensections stored at −80° C. using AS-BI-naphthol-β-D-glucuronic acid(Sigma) as described in Wolfe and Sands (2000) and Ghodsi et al. (1998).

For detection of storage vacuoles, tissues were fixed in 2.5%glutaraldehyde in 0.1 N cacodylate buffer for at least 48 hours at 4° C.Tissues were embedded in Epon 812 resin (Electron Microscopy Sciences,Ft. Washington, Pa.). Sections (0.5 μm) were prepared and stained withtoluidene blue as described in Wolfe and Sands (2000).

Results

The transposon plasmid carried human GUSB cDNA regulated by a strongpromoter (FIG. 6) which expressed very high levels of β-glucuronidase.Correct assembly of pT-CAGG5-GUSB was validated by restriction enzymeanalysis, and the structure of GUSB was confirmed by sequencing (datanot shown). GUSB expression was confirmed by transfection of primaryβ-glucuronidase-deficient murine fibroblasts. To determine whetherbeta-glucuronidase can be detected in plasma 48 hours after hydrodynamicinfusion of adult MPS VII mice and wild-type mice. Mice at 8-12 weeks ofage were injected with 25 μg of either pT-CAGG5-GUS plasmid orpBluescript. All mice tolerated the procedure well with only one deathof a wild-type mouse.

The plasmid pT-CAGGS-GUS mediated high levels β-glucuronidase expressionin the liver after hydrodynamic infusion. In GUS-deficient mice, as wellas in wild-type mice, foci of intensively bright red staining wereevenly distributed in liver tissue (FIG. 7). Counterstaining with methylgreen permitted localization GUSB activity to hepatocytes(predominantly) and Kupffer cells. Varying degrees of staining wereobserved in all cells, suggesting enzyme cross-correction.Beta-glucuronidase activity (Table 4) in treated MPS VII animalsexceeded that in untreated wild-type mice by over 10-fold.

TABLE 4 Beta-glucuronidase Enzyme Activity in Plasma and Liver 48-hoursafter Hydrodynamic Infusion of pT-CAGGS-GUSB plasmid. Enzyme activities*MICE Injection GUS hexosaminidase galactosidase §Gen Weight Vol. TimeQuality** Liver Plasma Liver Plasma Liver PT/CAGGS-GUS, 25 μg MPSVII 1 M25.0 g 2.5 ml 10 seconds Excellent 2,552 5,209 4,300 5890 124 MPSVII 2 M24.7 g 2.5 ml 12 seconds Good 1,860 2,715 4,386 N/A 132 MPSVII 3 M 22.9g 2.3 ml 19 seconds Poor 99 3 5,185 1,229 123 MPSVII 4 F 19.0 g 1.9 ml12 seconds Fair 2 2 6,561 8,107 123 Unaffected 5 F 20.3 g 2.0 ml 10seconds Good 75 119 936 953 49 Unaffected 6 M 30.6 g 3.0 ml 14 secondsFair 16 123 675 611 43 Unaffected 7∞F 19.9 g 2.0 ml 10 seconds Excellent314 297 911 453 49 Unaffected 8 M 26.8 g 2.7 ml  9 seconds ExcellentDied within 1 hour post injection pBluescript, 25 μg MPSVII 9 F 20.4 g2.0 ml Injection failed, mouse survived but was not used further MPSVII10 F 17.7 g 1.8 ml 10 seconds Excellent 1 2 6,786 1,365 130 MPSVII 11 F21.4 g 2.1 ml  8 seconds Excellent 1 1 6,526 4,750 153 MPSVII 12 F 20.0g 2.0 ml  8 seconds Excellent 1 2 7,634 1,520 179 Unaffected 13 M 28.6 g2.9 ml 15 seconds Poor 118 7 696 491 42 Unaffected 14 F 28.0 g 2.8 ml  9seconds Excellent 9 9 1,187 792 75 Unaffected 15 F 22.9 g 2.3 ml 12seconds Poor 86 9 845 633 38 Unaffected 16 F 22.8 g 2.3 ml  9 secondsExcellent 94 12 1,031 649 37 Untreated MPSVII 17 M — — — — 1 1.7 5,2101,365 125 MPSVII 18 F — — — — 1.3 1.4 7,231 2,679 148 MPSVII 19 F — — —— 1.1 1.9 6,718 1,868 130 Unaffected 20 F — — — — 87 14 985 675 44Unaffected 21 F — — — — 84 9.6 1,004 ? ? Unaffected 22 F — — — — 188 15685 589 42 *Enzyme activity is expressed as nmoles of 4 MU/mg protein/hfor liver and as nmoles of 4 MU/ml plasma/h for plasma §Gen stands forgender and genotype **Time and Quality refer to the microinjection timeand apparent success at the time of injection ∞This mouse is areplacement for an unaffected mouse, in which injection failed (theneedle did not go into the vein)

β-glucuronidase activity was easily detectable in plasma (Table 4), andmay be used as a presumptive indicator of the presence/absence ofpT-CAGGS-GUSB. This proved to be a convenient method of monitoring thesuccess of the procedure for administering the test agent.

Eight-week β-glucuronidase enzyme activity in plasma after co-injectionwith SB plasmid. MPS VII mice were either injected with pT/CAGGS-GUSBalone (Treatment Group 1) or co-injected with pCMV-SB10 at two differentmolar ratios of the transposon to transposase plasmid: 1:1 (TreatmentGroup 2) and 10:1 transposon to transposase plasmid (Treatment Group 3).All treated mice received an equal amount of 25 μg of pT/BH-CAGGS-GUS.The injected DNA amount was kept equal in all mice by using pBluescriptas the filler plasmid. MPS VII mice from the negative control groupreceived pBluescript alone. Forty-eight hours after injection,β-glucuronidase activities in the plasma of treated animals were equallyhigh in all three surviving animals and in the same range as those inthe short-term experiment (Table 5). One week post-injection, plasmaβ-glucuronidase in mice from Treatment Group 1 was reduced to 71.8% ofinitial 48 hour post injection activity. In Treatment Groups 2 and 3,enzymatic activity had decreased to 37.3% and 28.6% of initial activity,respectively. At one month, β-glucuronidase in plasma was virtuallyundetectable in all three groups. Secondary elevations of otherlysosomal enzymes concomitant with the deficiency of the causativeenzyme have been observed in storage diseases and respond to treatment.In this series of mice, hexosaminidase levels in untreated mice werefound to be pathologically elevated. At time point 1 week,hexosaminidase levels in plasma decreased to 78.5% in Treatment Group 1;58.5% in Treatment Group 2; and 61.8% in Treatment Group 3 as comparedto time-point 2 days post-injection. No reduction of hexosaminidaseactivity was observed in sham-treated MPS VII mice. At a later timepoint (time point, weeks/hours), levels remained/changed (Table 5).

TABLE 5 Beta-glucuronidase Activity in Plasma 2 and 7 days afterco-injection by hydrodynamic infusion of pT-CAGGS-GUS plasmid % Initial% initial 2 days 7 days Activity Mouse 2 Days 7 Days Activity HEX inplasma, nmole/ml/hr pT/CAGGS_GUS 1 5304 3855 72.7 3,300 2160 65.5 3 36022114 58.7 3492 1980 56.7 4 2002 1868 93.3 2211 113.4 Mean 3636 ± 16512612 ± 1083 74.9 ± 17.4%, 3001 ± 691 2216 ± 268 n = 3 pT/CAGGS-GUSB +pSB10 1:1 5 2692 566 21 3491 2132 61.1 6 3800 823 21.7 6600 2165 32.8 76405 2074 32.4 2376 2039 85.8 8 3119 1116 35.8 3432 1855 54.1 Mean 40041145 28.6%, n = 4 3975 2048 58.5 pTCAGGS-GUSB plus pSB10, 10:1 9 77112565 33.3 2343 1835 78.3 10  3372 1172 34.8 2970 1835 61.8 11  0 0 79463610 45.4 12  1552 982 63.3 3928 N/A Mean 4212 1573 37.3%, n = 3 44202427 61.8 pBluescript 13  Neg. control 0 2897 3274 113 14  Neg. control0 3815 N/A Wild-type 6.9-14.9 (n = 6) Untreated

Eight-week enzyme levels in organs. Two months after administration ofpTCAGGS-GUSB transposon, beta-glucuronidase persisted in liver andspleen in all three groups and the levels of β-glucurorudase in animalsthat did not receive transposase were higher than in those that wereco-injected with the transposase plasmid. Histochemical stainingrevealed considerable reduction of positively stained cells both inliver and in spleen (FIG. 8). Fluorometric quantification ofβ-glucuronidase activity showed that in Treatment Group 1 it wasapproximately 8-fold higher than in either group 1 or 2 (Table 7).

TABLE 6 Beta-glucuronidase activity in MPS VII mouse organs 1 week and 1month after injection, nmol/mg protein/hr Mouse Organ #2 (No SB) #14(SB1:10) #8 (SB1:1) W.T. Liver 5184 6185 28 119-188 n = 6 Spleen 60804534 3.9 269-290 n = 3 Heart 98 94 2.1 10-13 n = 3 Kidney 59 59 1.960-74 n = 3 Lung 49 65 1.8 60-83 n = 3 Gonad N/A N/A 0 223 n = 1(testis) (testis) (ovary) Brain N/A N/A 0 17-20 n = 3

TABLE 7 GUSB activity in liver and spleen 8 weeks post-injection,nmol/mg/hr Transposon pT- CAGGS- Transposase Inert DNA β-glucuronidaseTreatment GUSB pCMV-SB10 pBluescript Liver Spleen Group (mcg) (mcg)(mcg) Mouse (nmol/mg/h) (nmol/mg/h) Transposon 25 0 12.5 1 29.5 2.1Alone 3 18.6 3.2 4 2.4 1.04 Transposon:Transposase 25 12.5 0 6 1.15 0.591:1 9 2.21 1.0 10 1.92 0.85 Transposon:Transposase 25 1.25 11.25 11 4.010.82 10:1 12 1.51 0.89 15 0.78 2.71 Control (−) 0 0 37.5 17 0.63 0.76 330.39 0.75 Control (+) Untreated 166.8; (119-188), 368; 269-290, n = 6 n= 3

Distribution of β-glucuronidase expression in various mouse organs wasstudied at one week (1 mouse from Treatment Group 1 and Treatment group3), 4 weeks (1 mouse from Treatment Group 2) (Table 6) and 8 weeks (n=3from each group) (Table 4). This study showed that during the first weekfollowing hydrodynamic-based administration, β-glucuronidase activitiesin liver and spleen were comparably high; the heart had levels less than2% that of liver and spleen. Moreover, the lung had less than 1%, andthe ovary had undetectable, β-glucuronidase activity.

Extent of correction of the pathology. No lesions were seen inHematoxylin and Eosin-stained 6 mm sections of liver, spleen, lung,testis, ovary, gut, cerebellum, kidney and heart from treated,sham-treated or untreated MPS mutant mice. Toluidene-blue staining of0.5 mm sections of liver and spleen revealed a dramatic reduction in thenumber and size of storage vacuoles in all treated groups (FIG. 8). InTreatment Group 1, where the sections were indistinguishable from thoseof normal controls, loss of storage vacuoles appeared complete. Partialreduction of storage was observed in Treatment Groups 2 and 3.Remarkably, storage vacuoles appeared to be not just prevented butactually eliminated in some mice (e.g., mouse #3 was 215 days of agewhen killed but had no vacuoles; the wt untreated control had manyvacuoles at 50 days of age. If treatment of #3 started after it was 50days of age, the data suggests that vacuoles were not just prevented,but actually eliminated).

Discussion

Therapeutic gene transfer and expression is widely held to require someextraneous mechanism(s) for molecular stabilization and for transmissionacross the tissue and cellular membranes. Thus, the most feasible formsof gene therapy have used extensively modified replication-defectiveviral vectors, liposomes or even ex vivo electroporation of DNA intocells prior to transplantation.

While studying a potential means of intravenous injection of unmodifiedDNA into mice, surprisingly it was found that hydrodynamicadministration of a particular plasmid structure was capable oflong-term expression with high levels of enzyme expression, achieving apotentially curative response, in a murine model ofmucopolysaccharidosis type VII. In particular, expression of the GUSBtransgene was easily detected in plasma. Notably, plasma levels ofglucuronidase were exceedingly high 1 week post-injection, but becamebarely detectable after 1 month. This suggests that such transientexpression was due to episomal delivery, and that the majority ofpT-CAGGS-GUSB plasmid did not persist in an integrated form. By 8 weekspost-injection, β-glucuronidase activity was undetectable in plasma;however, glucuronidase activity was detectable in the liver and spleen.Beta-glucuronidase activity in two out of three mice in Treatment Group1 was over 10% that of wt, whereas in Treatment Groups 2 and 3 theselevels were around 1% that of normal values. The levels observed weresufficient to result in the first successful treatment of a metabolicdisease by intravenous injection of a plasmid.

Further, the results show there was a “dose effect”, with levels ofexpression corresponding to the level of reversal of pathology. Theselevels of expression were sufficient to reverse accumulation of GAG.

As observed by the correlation of glucuronidase enzyme activity tocorrection (e.g., FIGS. 7 and 8, and Table 7), increases in enzymeactivity in the liver corresponded to the degree of metaboliccorrection. Animals that had the highest level of glucuronidase activity8 weeks after treatment were clear of pathologic lysosomalaccumulations. In these animals, there appears to be a cure of metabolicdisease. By extension to the work of enzyme replacement in this sameanimal model (Sands et al., 1997), this would correspond to a cure ofthe murine MPS VII phenotype in other studies, especially usingnon-viral gene transfer systems.

The remarkable aspect of the reported study is the apparent ability tocure this storage disease with out the necessity of using a viralvector. This is the first time a long-term effect has resulted fromnaked DNA gene therapy (i.e., without the benefit of a viral vector).

Interestingly, beta-glucuronidase levels were lower when transposase waspresent. For example, eight-weeks after pT-CAGGS-GU5B administration,glucuronidase expression in Treatment Group 1 was higher than in any(p<0.01) or all (p<0.01) of the others (Treatment Group 2 and TreatmentGroup 3). This difference was not random, but was statisticallysignificant. Notably, mice Treatment Group 2 and Treatment Group 3received the same amount of pT-CAGGS-GUSB, but showed much lower levelsof glucuronidase expression (p<0.01). From this observation, SBtransposase may be responsible for these lower levels.

Based on these observations, it is likely that expression ofglucuronidase activity is predominantly from an episomal form.Nevertheless, it appears that episomal gene expression by this means hasthe potential for effecting a long-term treatment.

TABLE 8 Dose effect of β-glucuronidase activity on storage clearance2-Day 8-Week 8-Week Liver Spleen Treatment Glucuronidase GlucuronidaseGlucuronidase Vacuole Vacuole Mb Group Activity Activity Liver ActivitySpleen Area Area 34 W.T. 14 166.8 368 0.18 0.09 1 pT- 5304 29.5 2.1 0.752.19 3 CAGGS- 3602 18.6 3.2 0.35 2.75 4 GUSB 2002 2.4 1.04 1.11 1.76 6pT- 2692 1.15 0.59 2.02 2.83 9 CAGGS- 6405 2.21 1.0 3.13 3.35 10 GUSB +3119 1.92 0.85 2.36 1.93 pCMV- SB10, 1:1 11 pT- 7711 4.01 0.82 1.03 1.8312 CAGGS- 3372 1.51 0.89 1.55 6.15 15 GUS + 1552 0.78 2.71 1.32 4.69pCMV- SB10, 10:1 13 Fail 0 0.26 0.52 9.13 11.85 Treatment 17 Sham- 00.63 0.76 13.65 N/A treated MPS 33 Untreated 0 .039 0.75 9.54 6.97 MPS

REFERENCES

-   Albritton et al., Cell, 57:659 (1989).-   Amado et al., Science, 285:674 (1999).-   Banerji et al., Cell, 33:729 (1983).-   Barranger et al., Japanese J. of Inher. Met. Disease, 51:45 (1989).-   Becker et al., Hum. Gene Ther., 10:2559 (1999).-   Blomer et al., J. Virol., 71:6641 (1997).-   Boshart et al., Cell, 41:521 (1985).-   Burns et al., Proc. Natl. Acad. Sci. USA, 90:8033 (1993).-   Byrne et al., Proc. Natl. Acad. Sci. U.S.A., 86:5473 (1989).-   Calame et al., Adv. Immunol., 43:235 (1988).-   Campes et al., Genes Dev., 3:537 (1989).-   Carlesso et al., Oncogene, 9:149 (1994).-   Case et al., Proc. Natl. Acad. Sci. USA, 96:2988 (1999).-   Cech, J. Amer. Med. Assn., 260:3030 (1988).-   Chang et al., Gene Ther., 6:715 (1999).-   Coll, Arch. Virol., 140:827 (1995).-   Costello et al., Gene Ther., 7:596 (2000).-   Douglas et al., Hum. Gene Ther., 10:935 (1999).-   Dull et al., J. Virol., 72:8463 (1998).-   Edlunch et al., Science, 230:912 (1985).-   Erickson et al., J. Biol. Chem., 260:14319 (1985).-   Fields et al., New Engl. J. Med., 277:989 (1967).-   Firon et al., Am. J. Hum. Genet., 46:527 (1990).-   Food and Drug Administration (1991)    http://www.fda.gov/cber/gdlns/ptcsomat.pdf.-   Food and Drug Administration (1998)    http://www.fda.gov/cber/gdlns/somgene.pdf.-   Furbish et al., Biochem. Biophys. Acta., 673:425 (1981).-   Ghidsi et al., Hum. Gene Ther., 9:2331 (1998).-   Goldman et al., Hum. Gene Ther., 8:2261 (1997).-   Gordon, Mol. Med. Today, 4:468 (1998).-   Gossen et al., Proc. Natl. Acad. Sci., 89:5547 (1992).-   Grosveld et al., Cell, 51:975 (1987).-   Haas et al., Mol. Ther., 2:71 (2000).-   Hackett et al., WO98/40510-   Heid et al., Genome Res., 6:986 (1996).-   Helene, Anticancer Drug Dis., 6(6):569 (1991.-   Ho et al., Proc. Natl. Acad. Sci. USA, 68:2810 (1971).-   Ivics et al., Cell, 91:501 (1997).-   Johnson et al., Gene Ther., 7:568 (2000).-   Kafri et al., J. Virol., 73:576 (1999).-   Kafri et al., Nat. Genet., 17:314 (1997).-   Kessel et al., Science, 249:374 (1990).-   Kim et al., J. Virol., 72:811 (1998).-   Konieczko et al., J. Virol., 199:200 (1994).-   Lever, Curr. Opin. Mol. Ther., 2:488 (2000).-   Marcus-Sakura, Anal. Biochem., 172:289 (1988).-   Maher et al., Antisense Res and Dev., 1:227 (1991)-   Marthas et al., J. Virol., 67:6047 (1993)-   Mastromarino et al., J. Gen. Virol., 68:2359 (1987).-   McKnight et al., Cell, 37:253 (1984).-   McNeish et al., Science, 241:837 (1988).-   Methods of Enzymology 65:499 (1980).-   Miyoshi et al., J. Virol., 72:8150 (1998).-   Miyoshi et al., Proc. Natl. Acad. Sci. USA, 94:10319 (1997).-   Miyoshi et al., Science, 283:682 (1999).-   Naldini et al., Proc. Natl. Acad. Sci. USA, 93:11382 (1996).-   Naldini et al., Science, 272:263 (1996).-   Neural Grafting in the Mammalian CNS, Bjorklund & Stenevi, eds.    (1985).-   Ng et al., Mol. Cell. Biol., 5:2720 (1985).-   O′Brian et al., Science, 241:1098 (1988).-   Orlando et al., Clin. Chem. Lab. Med., 36:255 (1998).-   Ory et al., Proc. Natl. Acad. Sci. 93:11400 (1996).-   Pan et al., Mol. Ther., 6:19 (2002).-   Park et al., Nat. Genet., 24:49 (2000).-   Pilaro et al., Toxicol. Pathol. 27:4 (1999).-   Pilling, Toxicol. Pathol., 27:678 (1999).-   Pinkert et al., Genes Dev., 1:268 (1987).-   Podsakoff, Mol. Ther., 4:282 (2001).-   Poeschla et al., Nat. Med., 4:354 (1998).-   Queen and Blatimore, Cell, 33:741 (1983).-   Rappeport et al., Birth Defects: Original Article Series 22, 1:101    (1986).-   Reiser et al., Proc. Natl. Acad. Sci. USA, 93:15266 (1996).-   Richter et al., Int. J. Hematol., 73:162 (2001).-   Romano et al., Stem Cells, 18:19 (2000).-   Sambrook et al., in Molecular Cloning: A Laboratory Manual, Cold    Spring Harbor Laboratory, N.Y. (1989).-   Sands et al., Neuro. Dis., 7:352 (1997)-   Sandhoff et al., In: The Metabolic Basis of Inherited Disease,    Scriver et al. (eds), McGraw-Hill, NY, pp. 1807-1839 (1989).-   Sattentau et al., Science, 234:1120 (1986).-   Scherr et al., BioTechniques, 31:520 (2001).-   Schnitzlein et al., J. Virol., 142:426 (1985).-   Schroers et al., Mol. Ther., 1:171 (2000).-   Takasaki et al., J. Biol. Chem., 259:10112 (1984).-   Trono., J. Virol., 66:4893 (1992).-   Tsuji et al., Proc. Natl. Acad. Sci. USA, 85:2349 (1988).-   Verdier et al., Toxicol. Sci., 47:9 (1999).-   Weintraub, Sci. Am., 262:40 (1990).-   Whitley et al., Birth Defects, 22:7 (1986).-   Winoto et al., EMBO J., 8:729 (1989).-   Wolff et al., Mol. Thera., 2:552 (2000).-   Woychik et al., Nature, 318:36 (1985).-   Woods et al., J. Intern. Med., 249:339 (2001).-   Xu et al., Mol. Ther., 5:141 (2002).-   Zhang et al., Human Gene Therapy. 10:1735 (1999).-   Zheng et al. Am. J. Hum. Genet., 69:679 (2001).-   Zufferey et al., Nat. Biotech, 15:871 (1997).

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

1. A method to prevent, inhibit or treat a disorder characterized by theabsence or reduced levels of a lysosomal enzyme in a mammal, comprising:administering to a vascular compartment of a mammal having or at risk ofthe disorder, an effective amount of a recombinant lentivirus comprisinga nucleic acid segment encoding the enzyme.
 2. (canceled)
 3. The methodof claim 1 wherein the protein is alpha-L-iduronidase,iduronate-2-sulfatase, heparan sulfate sulfatase,N-acetyl-alpha-D-glucosaminidase, beta-hexosamine, alpha-galactosidase,beta-galactosidase, beta-glucuronidase or glucocerebrosidase.
 4. Themethod of claim 1 wherein the recombinant lentivirus comprises aheterologous promoter operably linked to the nucleic acid segment. 5.The method of claim 1 wherein vascular compartment is a vein, artery,bone marrow cavity, heart, spleen, umbilical cord vessel or placenta. 6.The method of claim 1 wherein the mammal is a human.
 7. (canceled) 8.The method of claim 1 wherein the recombinant lentivirus is apseudotyped virus.
 9. The method of claim 1 wherein the lentivirus is ahuman immunodeficiency virus-1 (HIV-1).
 10. The method of claim 1wherein the disorder is a mucopolysaccharide disorder.
 11. The method ofclaim 10 wherein the disorder is a mucopolysaccharidosis type Idisorder.
 12. The method of claim 10 wherein the disorder is amucopolysaccharidosis type VII disorder.
 13. (canceled)
 14. Therecombinant virus of claim 18 wherein the enzyme is alpha-L-iduronidase,beta-hexosamine, alpha-galactosidase, beta-galactosidase,beta-glucuronidase or glucocerebrosidase.
 15. The recombinant virus ofclaim 18 wherein the lentivirus is HIV-1.
 16. The recombinant virus ofclaim 18 wherein the U3 of the 5′LTR is modified by substantiallyreplacing the U3 of the 5′LTR with a heterologous promoter.
 17. Therecombinant virus of claim 18 which further comprises a promoteroperably linked to the nucleic acid segment.
 18. Recombinant viruscomprising a lentivirus vector comprising a 5N LTR and a 3N LTR and anucleic acid segment encoding a lysosomal enzyme, wherein the U3 regionof at least one LTR is optionally modified.
 19. The recombinant virus ofclaim 18 which is a pseudotyped virus.
 20. A method for providing abiologically active lysosomal enzyme to a cell of a mammal, comprising:contacting the cell with an effective amount of a recombinant lentiviruscomprising a nucleic acid segment encoding the enzyme.
 21. The method ofclaim 1 wherein the mammal is a newborn.
 22. The method of claim 1wherein the lentivirus is intravenously administered.